In vivo production of cyclic peptides for inhibiting protein-protein interaction

ABSTRACT

The present invention relates to methods and compositions utilizing inteins to generate libraries of cyclic peptides in vivo. The prevent invention also relates to methods for inhibiting protein-protein interaction.

FIELD OF THE INVENTION

The present invention relates to methods and compositions for generatingintracellular cyclic peptide and protein libraries. In addition, thepresent invention relates to methods and compositions for inhibitingprotein-protein interaction.

BACKGROUND OF THE INVENTION

Combinatorial libraries of synthetic and natural products are importantsources of molecular information for the development of pharmacologicagents. Linear peptide libraries, containing known and random peptidesequences, are particularly good sources of new and novel compounds fordrug development because of the diversity of structures which can begenerated. Drawbacks to linear peptide libraries include the following:(1) linear peptides are generally flexible molecules with entropiclimitations on achieving productive biologically active conformations;(2) linear peptides are susceptible to proteolytic enzymes; and, (3)linear peptides are inherently instable. For this reason, approachesutilizing conformational and topographical constraints to restrict thenumber of conformational states a peptide molecule may assume have beensought. See, for example, Hruby, Life Sci., 31:189 (1982) and Hruby etal., Biochem. J. 268:249 (1990).

Head-to-tail (backbone) peptide cyclization has been used to rigidifystructure and improve in vivo stability of small bioactive peptides (seeCamarero and Muir, J. Am. Chem. Soc., 121:5597-5598 (1999)). Animportant consequence of peptide cyclization is retention of biologicalactivity and/or the identification of new classes of pharmacologicalagents. Cyclic peptides have been reported that inhibit T-cell adhesion(Jois et al., J. Pept. Res., 53:18-29 (1999)), PDGF action (Brennand etal., FEBS Lett., 413:70-74 (1997)), and function as new classes of drugs(Kimura et al., (1997) J. Antibiot., 50:373-378; Eriksson et al., Exp.Cell Res., 185:86-100 (1989)).

Strategies for the preparation of circular polypeptides from linearprecursors have been described. For example, a chemical cross-linkingapproach was used to prepare a backbone cyclized version of bovinepancreatic trypsin inhibitor (Goldenburg and Creighton, J. Mol. Biol.,165:407-413 (1983)). Other approaches include chemical (Camarero et al.,Angew. Chem. Int. Ed., 37:347-349 (1998); Tam and Lu, Prot. Sci.,7:1583-1592 (1998); Camarero and Muir, Chem: Commun., 1997:1369-1370(1997); and Zhang and Tam, J. Am. Chem. Soc. 119:2363-2370 (1997)) andenzymatic (Jackson et al., J. Am. Chem. Soc., 117:819-820 (1995))intramolecular ligation methods which allow linear synthetic peptides tobe efficiently cyclized under aqueous conditions. However, therequirement for synthetic peptide precursors has limited thesechemical/enzymatic cyclization approaches to systems that are both exvivo and limited to relatively small peptides.

One solution to this problem has been to generate circular recombinantpeptides and proteins using a native chemical ligation approach. Thisapproach utilizes inteins (internal proteins) to catalyze head-to-tailpeptide and protein ligation in vivo (see, for example, Evans et al., J.Biol. Chem. 274:18359-18363 (1999); Iwai and Plückthun, FEBS Lett.459:166-172 (1999); Wood et al., Nature Biotechnology 17:889-892 (1999);Camarero and Muir, J. Am. Chem. Soc. 121:5597-5598 (1999); and Scott etal., Proc. Natl. Acad. Sci. USA 96:13638-13643 (1999)).

Inteins are self-splicing proteins that occur as in-frame insertions inspecific host proteins. In a self-splicing reaction, inteins excisethemselves from a precursor protein, while the flanking regions, theexteins, become joined to restore host gene function. Inteins can alsocatalyze a trans-ligation self-splicing reaction. Approaches making useof the trans ligation reaction include splitting the intein into twoparts and reassembling the two parts in vitro, each fused to a differentextein (Southworth et al., EMBO J. 17:918-926 (1998)). A somewhatdifferent approach uses an intein domain, and the reaction is thentriggered with a thiolate nucleophile, such as DTT (Xu et al., ProteinSci. 7:2256-2264 (1998)).

The ability to construct intein fusions to proteins of interest hasfound several applications. For example, inteins can be used inconjunction with an affinity group to purify a desired protein (Wood etal., Nature Biotechnology, 17:889-892 (1999)). Circular recombinantfusion proteins have been created by cloning into a commerciallyavailable intein expression system (Camarero and Muir, J. Am. Chem. Soc.121:5597-5598 (1999); Iwai and Plückthun, FEBS Lett. 459:166-172 (1999);and Evans et al., J. Biol. Chem. 274:18359-18363 (1999)). In anotherapproach, a mechanism for in vivo split intein-mediated circularligation of peptides and proteins via permutation of the order ofelements in the fusion protein precursor has been used to express cyclicproducts in bacteria (Scott et al., Proc. Natl. Acad. Sci. USA,96:13638-13643 (1999)).

Cyclic peptide libraries have been generated in phage (Koivunen et al.,Biotechnology 13:265-70 (1995)) and by using the backbone cyclicproteinomimetic approach (Friedler et al., Biochemistry, 37:5616-22(1998)). Methods for modifying inteins for the purpose of creatingcyclic peptides and/or proteins have been recently described (Benzoic,et al., WO 00/36093). It is an object of this invention to utilizeintein function, derived from wild-type or mutant intein structures, togenerate cyclic peptide libraries in vivo. The utilization of mutantintein structures for this purpose are of particular focus since thesehave been optimized for function in the specific context of an inteinscaffold engineered to result in peptide/protein cyclization. Methodsare described for generating, identifying, and utilizing mutants withaltered splicing/cyclization activity for use with cyclicpeptide/protein libraries. Intein-generated cyclic libraries aredescribed for the identification of cyclic peptides/proteins capable ofaltering a given cellular phenotype. Accordingly, it is an object of theinvention to provide compositions and methods useful in the generationof random fusion polypeptide libraries in vivo and the use of suchpeptides.

SUMMARY OF THE INVENTION

In accordance with the objects outlined above, the present inventionprovides methods of identifying a cyclic peptide capable of preventinginteraction of a first and a second interacting protein. The methodcomprises providing a plurality of cells comprising a first and a secondinteracting protein and contacting the cells with a library of fusionnucleic acids, which nucleic acids comprise sequences encoding aC-terminal and an N-terminal intein motif and a peptide. In a preferredembodiment, the peptide is a random peptide. In another embodiment, thepeptide is a biased randomized peptide. The method finally comprisesidentifying a cell comprising a cyclic peptide capable of preventing theinteraction of the interacting proteins.

In preferred embodiments, the interacting proteins are ubiquitin agents,such as ubiquitin activating proteins, ubiquitin conjugating proteins,and ubiquitin ligating proteins. In some embodiments, the interactingproteins are ubiquitin moieties and ubiquitin substrates. The first andsecond interacting proteins are selected from the above protein types inpairs known or believed to be interacting proteins in the process ofubiquitination.

In preferred embodiments, the cell comprising the cyclic peptide capableof preventing the interaction of the interacting proteins is identifiedby way of a fluorescence interaction between fluorescent labels on theinteracting proteins or between a fluorescent label on one interactingprotein and a quencher on the other interacting protein. In otherembodiments, the cell comprising a cyclic peptide capable of preventingthe interaction of the interacting proteins is identified using atwo-hybrid system, such as a yeast two hybrid system or a mammaliantwo-hybrid system, either of which comprises expression of a reporter inresponse to the binding or interaction of two subunits of atranscription activating protein.

The present invention also provides a method comprising providing alibrary of cells each comprising first and second interacting proteins,contacting the cells with a fusion nucleic acid comprising from 5′ to 3′nucleic acid encoding a C-terminal intein motif, nucleic acid encoding apeptide and nucleic acid encoding an N-terminal intein motif underconditions whereby a cyclic peptide is formed, and determining theability of the cyclic peptide to prevent interaction of said interactingproteins.

In addition, the invention provides a method comprising contacting acell culture with an intein-catalyzed cyclic peptide library such thatcells in said culture receive not more than one member of said library,monitoring the cells for a change in ubiquitination of a molecule, andisolating the nucleic acid that encodes the cyclic peptide that whenintroduced into said cell caused said change in ubiquitination of saidmolecule.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A depicts head to tail protein cyclization byreconfigured/engineered intein.

FIG. 1B depicts the mechanism of cyclization by reconfigured/engineeredintein.

FIG. 2A depicts intein catalyzed ligation by the Mxe GyrA intein. In anormal configuration, intein catalyzed ligation joins the exteinresidues located at the junction points with each of the two inteinmotifs.

FIG. 2B depicts the outcome of a motif reorganization resulting in theproduction of a cyclic peptide. Motif reorganziation involves providingintein B with its own translational start codon and placing intein Bamino-terminal to intein A.

FIG. 3A depicts the amino acid sequence of intein Ssp DnaB fromSynechocystis spp. strain PCC6803.

FIG. 3B depicts the amino acid sequence of intein Mxe GyrA fromMycobacterium xenopi.

FIG. 3C depicts the amino acid sequence of intein Ceu ClpP fromChlamydomonas eugametos.

FIG. 3D depicts the amino acid sequence of intein CIV RIR1 from Chiloiridescent virus.

FIG. 3E depicts the amino acid sequence of intein Ctr VMA from Candidatropicalis.

FIG. 3F depicts the amino acid sequence of intein Gth DnaB fromGuillardia theta.

FIG. 3G depicts the amino acid sequence of intein Ppu DnaB from Porphyrapurpurea.

FIG. 3H depicts the amino acid sequence of intein Sce VMA fromSaccharomyces cerevisiae.

FIG. 3I depicts the amino acid sequence of intein Mf1 RecA fromMycobacterium flavescens.

FIG. 3J depicts the amino acid sequence of intein Ssp DnaE fromSynechocystis spp. strain PCC6803.

FIG. 3K depicts the amino acid sequence of intein Mle DnaB fromMycobacterium leprae.

FIG. 3L depicts the amino acid sequence of intein Mja KlbA fromMethanococcus jannaschii.

FIG. 3M depicts the amino acid sequence of intein Pfu KlbA fromPyrococcus furiosus.

FIG. 3N depicts the amino acid sequence of intein Mth RIR1 fromMethanobacterium thermoautotrophicum (delta H strain).

FIG. 3O depicts the amino acid sequence of intein Pfu RIR1-1 fromPyrococcus furiosus.

FIG. 3P depicts the amino acid sequence of intein Psp-GBD Pol fromPyrococcus spp. GB-D.

FIG. 3Q depicts the amino acid sequence of intein Thy Pol-2 fromThermococcus hydrothermalis.

FIG. 3R depicts the amino acid sequence of intein Pfu IF2 fromPyrococcus furiosus.

FIG. 3S depicts the amino acid sequence of intein Pho Lon fromPyrococcus horikoshii OT3.

FIG. 3T depicts the amino acid sequence of intein Mja r-Gyr fromMethanococcus jannaschii.

FIG. 3U depicts the amino acid sequence of intein Pho RFC fromPyrococcus horikoshii OT3.

FIG. 3V depicts the amino acid sequence of intein Pab RFC-2 fromPyrococcus abyssi.

FIG. 3W depicts the amino acid sequence of intein Mja RtcB (Mja Hyp-2)from Methanococcus jannaschii.

FIG. 3X depicts the amino acid sequence of intein Pho VMA fromPyrococcus horikoshii OT3.

FIG. 4A depicts the amino acid sequence of a modified wild-type Ssp DnaBIntein configured to generate a cyclic peptide containing a FLAGepitope. The DNA sequence is given in FIG. 4B.

FIGS. 5A and B depict the nucleotide and amino acid sequence of theintein Ssp DnaB J3 template used to generate intein mutants L7-J3,E6-J3, E9-J3, C11-J3 and B8-J3, with improved splicing efficiency. TheJ3 template carries a mutation which results in an amino acid change Dto N at position 320. Thus, all mutants based on the J3 template aredouble mutants.

FIGS. 5C and D depict the nucleotide and amino acid sequence of inteinmutant L7-J3. L7 has two mutations which result in amino acidchanges: 1) D to N at position 320 and 2) R to K at position 389.

FIGS. 5E and F depict the nucleotide and amino acid sequence of inteinmutant E6-J3. E6 has two mutations which result in amino acidchanges: 1) D to N at position 320 and 2) I to V at position 34.

FIGS. 5G and H depict the nucleotide and amino acid sequence of inteinmutant E9-J3. E9 has two mutations which result in amino acidchanges: 1) D to N at position 320 and 2) T to A at position 36.

FIGS. 51 and J depict the nucleotide and amino acid sequence of inteinmutant C1-J3. C11 has two mutations which result in amino acidchanges: 1) D to N at position 320 and 2) S to P at position 23.

FIGS. 5K and L depict the nucleotide and amino acid sequence of inteinmutant B8-J3. B8 has two mutations which result in amino acidchanges: 1) D to N at position 320 and 2) K to R at position 369.

FIGS. 5M and N depict the nucleotide and amino acid sequence of inteinmutant L7-wt, which was generated from an Ssp DnaB wild-type (wt)template. Mutants generated from the wt template carry a single mutationwhich alters splicing efficiency. L7-wt carries a single mutation whichresults in the amino acid change R to K at position 389.

FIGS. 50 and P depict the nucleotide and amino acid sequence of inteinmutant C11-wt. C11-wt has a single mutation which results in the aminoacid change S to P at position 23.

FIGS. 5Q and R depict the nucleotide and amino acid sequence of inteinmutant E6-wt. E6-wt has a single mutation which result in the amino acidchange I to V at position 34.

FIG. 6 depicts the DNA sequence for a N-terminally fused GFP version ofthe Ssp DnaB intein.

FIG. 7 depicts reporter proteins which can be used for the selectionand/or detection of intein-based libraries.

FIG. 8 depicts localization sequences which can be used to target cyclicpeptide libraries intracellularly or extracellulary.

FIG. 9 depicts a random mutagenesis approach used in the optimization ofintein cyclization function.

FIG. 10 depicts a biotinylation approach for use in a yeast two hybridsystem.

FIG. 11 depicts a single chain antibody (SCAb) approach for use in ayeast two hybrid system.

FIG. 12 depicts the fluorescent reporter system used to quantify inteincyclization. FIG. 12A depicts GFP split at the loop 3 junction andreversal of the translation order of the N— and C-terminal fragments.The termini are fused using a glycine-serine linker. The GFP ispositioned within the Ssp DnaB intein cyclization scaffold. Cyclizedproduct reconstitutes both structure and fluorescence of GFP. Inaddition, splicing one-half of the myc epitope onto either side of theloop 3 junction allows for reconstruction of the myc epitope uponcyclization.

FIG. 12B shows the amino acid sequence of DNAB intein cyclizationscaffold with GFP.

FIG. 12C depicts the mechanism of intein catalyzed cyclization ofinverted loop 3 of GFP.

FIG. 12D shows the results from a FACS analysis of the cyclizationefficiency of wild-type Ssp DnaB intein in mammalian cells.

FIG. 12E shows the results from a Western analysis of a Ssp DnaBcatalyzed cyclization in mammalian cells.

FIG. 12F shows the results from a native gel and the signals from GFPfluorescence. The majority of the fluorescence arises from the formationof cyclized GFP product, bands C and D.

FIG. 13 illustrates a functional screen for isolating randomly-generatedmutants with altered cyclization activity. FIG. 13A depicts a functionalscreen for intein mutants with altered cyclization activity. FIG. 13Bdepicts mutations modeled on the Mxe GyrA intein structure. FIG. 13Cdepicts the sequence alignment of Mxe GyrA and Ssp DnaB inteins. Mutantsare identified in shaded color. FIG. 13D shows the results from awestern analysis of isolated mutants. DnaB mutants E9-J3, E6-J3, C11-J3,L7-J3, and B8-J3 have cyclization efficiencies were are greater than theJ3 starting intein template.

FIG. 14 depicts intein-mediated excision/ligation in mammalian cells.FIG. 14A depicts constructs in which Ssp DnaB intein is inserted intoloop 3 of GFP (i.e., GAB) or GFP with a C-terminal myc epitope. FIG. 14Bdepicts constructs similar to those shown in 14A, except that the mycepitope half-sites are positioned onto the extreme ends of each splicejunction. FIG. 14C depicts Western blot analysis of lysates fromtransfected Phoenix cells. Lanes 3 and 4 demonstrate efficient splicingwith only slight amounts of unspliced product detected.

FIGS. 15A-D depict a method for detecting cyclic peptides in mammaliancells. FIG. 15A depicts an overview of the method in which cyclicpeptides are detected in mammalian cells expressing a GFP fused inteinscaffold with cyclic peptide inserts. FIGS. 15B and C depict the MSanalysis of mammalian cell lysates expressing the cyclic peptideproducts from RGD7 (15B) and RGD9 (15C). FIG. 15D depicts an example ofLC/MS fragmentation fingerprinting of the cyclic peptide product of anintein construct.

FIG. 16 depicts the low energy conformers associated with cyclic peptideSRGDGWS.

FIG. 17 depicts the low energy conformers associated with cyclic peptideSRGPGWS.

FIG. 18 shows the amino acid sequence of human ubiquitin.

FIGS. 19A and 19B show the nucleic acid sequence and amino acidsequence, respectively, of a human E1, Uba1 (E1).

FIGS. 20A and 20B show the nucleic acid sequence and amino acidsequence, respectively, of a human E1, Uba3 homolog.

FIGS. 21A and 21B show the nucleic acid sequence and amino acidsequence, respectively, of a human E1, SAE1.

FIGS. 22A and 22B show the nucleic acid sequence and amino acidsequence, respectively, of a human E1, UBE1L.

FIGS. 23A and 23B show the nucleic acid sequence and amino acidsequence, respectively, of a human E1, APG7 isoform.

FIGS. 24A and 24B show the nucleic acid sequence and amino acidsequence, respectively, of a human E1, FLJ14657.

FIGS. 25A and 25B show the nucleic acid sequence and amino acidsequence, respectively, of a human E2, FTS.

FIGS. 26A and 26B show the nucleic acid sequence and amino acidsequence, respectively, of a human E2, XM_(—)054332.

FIGS. 27A and 27B show the nucleic acid sequence and amino acidsequence, respectively, of a human E2, Ubc8.

FIGS. 28A and 28B show the nucleic acid sequence and amino acidsequence, respectively, of a human E2, UbcH9.

FIGS. 29A and 29B show the nucleic acid sequence and amino acidsequence, respectively, of a human E2, Ubc12.

FIGS. 30A and 30B show the nucleic acid sequence and amino acidsequence, respectively, of a human E2, MGC10481.

FIGS. 31A and 31B show the nucleic acid sequence and amino acidsequence, respectively, of a human E2, UbcH6.

FIGS. 32A and 32B show the nucleic acid sequence and amino acidsequence, respectively, of a human E2, HIP2.

FIGS. 33A and 33B show the nucleic acid sequence and amino acidsequence, respectively, of a human E2, Uev1.

FIGS. 34A and 34B show the nucleic acid sequence and amino acidsequence, respectively, of a human E2, Ubc13.

FIGS. 35A and 35B show the nucleic acid sequence and amino acidsequence, respectively, of a human E3, MDM2.

DETAILED DESCRIPTION OF THE INVENTION

Peptide libraries are an important source of new and novel drugs.However, a number of hurdles must be overcome in order to express andsubsequently screen functional peptides and proteins in cells. Foremostamong these hurdles is the need to retain biological activity of thepeptides in a cellular environment. To overcome this problem, thepresent invention is directed to fusions of intein motifs and randompeptides such that circular peptides are formed which retain biologicalactivity.

Thus, generally, the present invention provides methods for generatinglibraries of cyclic peptides using inteins. Inteins are self-splicingproteins that occur as in-frame insertions in specific host proteins. Ina self-splicing reaction, inteins excise themselves from a precursorprotein, while the flanking regions, the exteins, become joined via anew peptide bond to form a linear protein. By changing the N to Cterminal orientation of the intein segments, the ends of the extein jointo form a cyclized extein. FIG. 1 illustrates intein catalyzed joiningof extein residues located at the junction points with each of the twointein motifs.

In addition, the invention provides methods for identifying cyclicpeptides that prevent or alter the interaction of interacting proteinsand the use of such peptides. Generally, the method includes providing acyclic peptide formed from an intein catalyzed reaction. Preferably, thecyclic peptide alters association of interacting proteins. In addition,the invention provides identifying the cyclic peptide that modulatesinteraction of interacting proteins. The cyclic peptide finds use as adrug or drug mimetic. Preferably, the cyclic peptide causes analteration in ubiquitination of a molecule.

Because intein function is not strongly influenced by the nature of theextein polypeptide sequences located between them, standard recombinantmethods can be used to insert random libraries into this position.Placement of these intein libraries into any number of delivery systemsallows for the subsequent expression of unique cyclic peptides withinindividual cells. Such cells can then be screened to identify peptidesof interest.

Accordingly, the present invention provides fusion polypeptidescomprising intein motifs and peptides.

By “fusion polypeptide” or “fusion peptide” or grammatical equivalentsherein is meant a protein composed of a plurality of protein components,that while typically unjoined in their native state, are joined by theirrespective amino and carboxyl termini through a peptide linkage to forma single continuous polypeptide. “Protein” in this context includesproteins, polypeptides and peptides.

Plurality in this context means at least two, and preferred embodimentsgenerally utilize two components. It will be appreciated that theprotein components can be joined directly or joined through a peptidelinker/spacer as outlined below. In addition, as outlined below,additional components such as fusion partners including targetingsequences, etc. may be used.

The present invention provides fusion proteins of intein motifs andrandom peptides. By “inteins”, or “intein motifs”, or “intein domains”,or grammatical equivalents herein is meant a protein sequence which,during protein splicing, is excised from a protein precursor. Alsoincluded within in the definition of intein motifs are DNA sequencesencoding inteins and mini-inteins.

Many inteins are bifunctional proteins mediating both protein splicingand DNA cleavage. Such elements consist of a protein splicing domaininterrupted by an endonuclease domain. Because endonuclease activity isnot required for protein splicing, mini-inteins with accurate splicingactivity can be generated by deletion of this central domain (Wood etal., Nature Biotechnology, 17:889-892 (1999); hereby incorporated byreference).

Protein splicing involves four nucleophilic displacements by threeconserved splice junction residues. These residues, located near theintein/extein junctions, include the initial cysteine, serine, orthreonine of the intein, which intiates splicing with an acyl shift. Theconserved cysteine, serine, or threonine of the extein, which ligatesthe exteins through nucleophilic attack, and the conserved C-terminalhistidine and asparagine of the intein, which releases the intein fromthe ligated exteins through succinimide formation. See Wood, et al.,supra.

Inteins also catalyze a trans-ligation reaction. The ability of inteinfunction to be reconstituted in trans by spatially separated inteindomains suggests that reorganization of the self-splicing motifs can beused to produce peptides with a circular topology.

In a preferred embodiment, the translational order in which the N— andC-terminal intein motifs are normally synthesized within a polypetidechain is reversed. Generally, a reversal in the translational order inwhich the N— and C-terminal intein motifs are synthesized should notfundamentally change the enzymatic function of the intein. However, thelocation of the intervening peptide's amino and carboxy termini arealtered in such a way that the product of the intein ligation reactionis no longer linear, but rather is cyclized. FIG. 2 illustrates theoutcome of a motif reorganization in which intein B has been given itsown translational start codon and placed amino-terminal to intein A. Toeffectively express unique peptides in cells, fusion polypetidescomprising a C-terminal motif, a peptide and a N-terminal motif areselected or designed for the production of random libraries of cyclicpeptides in vivo.

In a preferred embodiment, the fusion polypeptide is designed with theprimary sequence from the N-terminus comprising I_(A)-target-I_(B).I_(A) is defined herein as the C-terminal intein motif, I_(B) is definedherein as the N-terminal intein motif and target is defined herein as apeptide. DNA sequences encoding the inteins may be obtained from aprokaryotic DNA sequence, such as a bacterial DNA sequence, or aeukaryotic DNA sequence, such as a yeast DNA sequence. The InteinRegistry includes a list of all experimental and theoretical inteinsdiscovered to date and submitted to the registry(www.neb.com/inteins/int_reg.html).

In a preferred embodiment, fusion polypeptides are designed using inteinmotifs selected from organisms belonging to the Eucarya and Eubacteria,with the intein Ssp DnaB (GenBank accession number Q55418) beingparticularly preferred. The GenBank accession numbers for other inteinproteins and nucleic acids include, but are not limited to: Ceu ClpP(GenBank acession number P42379); CIV RIR1 (T03053); Ctr VMA (GenBankaccession number A46080); Gth DnaB (GenBank accession number O78411);Ppu DnaB (GenBank accession number P51333); Sce VMA (GenBank accessionnumber PXBYVA); Mf1 RecA (GenBank accession number not given); Mxe GyrA(GenBank accession number P72065); Ssp DnaE (GenBank accession numberS76958 & S75328); and Mle DnaB (GenBank accession number CM17948.1)

In other embodiments, inteins with alternative splicing mechanisms arepreferred (see Southworth et al., EMBO J., 19:5019-26 (2000)). TheGenBank accession numbers for inteins with alternative splicingmechanisms include, but are not limited to, Mja KlbA (GenBank accessionnumber Q58191) and Pfu KlbA (PF_(—)949263 in UMBI).

In yet other embodiments, inteins from thermophilic organisms are used.Random mutagenesis or directed evolution (e.g., PCR shuffling, etc.) ofinteins from these organisms could lead to the isolation of temperaturesensitive mutants. Thus, inteins from thermophiles (e.g., Archaea) whichfind use in the invention are: Mth RIR1 (GenBank accession numberG69186); Pfu RIR1-1 (AAB36947.1); Psp-GBD Pol (GenBank accession numberAAA67132.1); Thy Pol-2 (GenBank accession number CAC18555.1); Pfu IF2(PF_(—)1088001 in UMBI); Pho Lon Baa29538.1); Mja r-Gyr (GenBankaccession number G64488); Pho RFC (GenBank accession number F71231); PabRFC-2 (GenBank accession number C75198); Mja RtcB (also referred to asMja Hyp-2; GenBank accession number Q58095); and Pho VMA (NT01PH1971 inTigr).

Preferred fusion polypeptides of the invention increase the efficiencyof the cyclization reaction by selecting or designing intein motifs withaltered cyclization activity when expressed in vivo. In a preferredembodiment, the fusion polypeptides of the invention employ the DNAsequence encoding the Synechocystis ssp. strain PCC6803 DnaB intein. Aparticularly preferred fusion polypeptide structure is illustrated inFIGS. 4A and 4B.

In a preferred embodiment, fusion polypeptides are designed using mutantintein sequences with altered cyclization activity as described below.Preferred mutant intein sequences include, but are not limited, to thoseshown in FIG. 5. In addition, preferred mutant intein sequences are setforth in U.S. patent application Ser. No. 10/197,927, filed Jul. 16,2002, which is expressly incorporated herein by reference.

In a preferred embodiment, the fusion polypeptides of the inventioncomprise peptides. That is, the fusion polypeptides of the invention aretranslation products of nucleic acids. In this embodiment, nucleic acidsare introduced into cells, and the cells express the nucleic acids toform peptides. Generally, peptides ranging from about 4 amino acids inlength to about 100 amino acids may be used, with peptides ranging fromabout 5 to about 50 being preferred, with from about 5 to about 30 beingparticularly preferred and from about 6 to about 20 being especiallypreferred.

In a preferred embodiment, the fusion polypeptides of the inventioncomprise random peptides. By “random peptides” herein is meant that eachpeptide consists of essentially random amino acids. Since generallythese random peptides (or nucleic acids, discussed below) are chemicallysynthesized, they may incorporate any amino acid at any position. Thesynthetic process can be designed to generate randomized proteins toallow the formation of all or most of the possible combinations over thelength of the sequence, thus forming a library of randomized peptides.

In a preferred embodiment, the fusion polypeptides of the inventioncomprise peptides derived from a cDNA library.

The fusion polypeptide preferably includes additional components,including, but not limited to, reporter proteins and fusion partners.

In a preferred embodiment, the fusion polypeptides of the inventioncomprise a reporter protein. By “reporter protein” or grammaticalequivalents herein is meant a protein that by its presence in or on acell or when secreted in the media allow the cell to be distinguishedfrom a cell that does not contain the reporter protein. As describedherein, the cell usually comprises a reporter gene that encodes thereporter protein.

Reporter genes fall into several classes, as outlined above, including,but not limited to, detection genes, indirectly detectable genes, andsurvival genes. See FIG. 6.

In a preferred embodiment, the reporter protein is a detectable protein.A “detectable protein” or “detection protein” (encoded by a detectableor detection gene) is a protein that can be used as a direct label; thatis, the protein is detectable (and preferably, a cell comprising thedetectable protein is detectable) without further manipulations orconstructs. As outlined herein, preferred embodiments of screeningutilize cell sorting (for example via FACS) to detect reporter (and thuspeptide library) expression. Thus, in this embodiment, the proteinproduct of the reporter gene itself can serve to distinguish cells thatare expressing the detectable gene. In this embodiment, suitabledetectable genes include those encoding autofluorescent proteins.

Detectable enzyme products resulting from the intein cyclizationreaction may also be used to detect cells that are expressing thedetectable product. Examples of enzymes which can be used includeluciferase, β-galactosidase, β-lactamase, puromycin resistance protein,etc.

As is known in the art, there are a variety of autofluorescent proteinsknown; these generally are based on the green fluorescent protein (GFP)from Aequorea and variants thereof; including, but not limited to, GFP,(Chalfie et al., “Green Fluorescent Protein as a Marker for GeneExpression,” Science 263(5148):802-805 (1994)); enhanced GFP (EGFP;Clontech—Genbank Accession Number U55762)), blue fluorescent protein(BFP; Quantum Biotechnologies, Inc., 1801 de Maisonneuve Blvd. West, 8thFloor, Montreal (Quebec) Canada H3H 1J9; Stauber, R. H., Biotechniques24(3):462-471 (1998); Heim, R. and Tsien, R. Y. Curr. Biol. 6:178-182(1996)), enhanced yellow fluorescent protein (EYFP; ClontechLaboratories, Inc., Palo Alto, Calif.) and red fluorescent protein. Inaddition, there are recent reports of autofluorescent proteins fromRenilla and Ptilosarcus species. See WO 92/15673; WO 95/07463; WO98/14605; WO 98/26277; WO 99/49019; U.S. Pat. No. 5,292,658; U.S Pat.No. 5,418,155; U.S. Pat. No. 5,683,888; U.S. Pat. No. 5,741,668; U.S.Pat. No. 5,777,079; U.S. Pat. No. 5,804,387; U.S. Pat. No. 5,874,304;U.S patent 5,876,995; and U.S. Pat. No. 5,925,558; all of which areexpressly incorporated herein by reference.

Preferred fluorescent molecules include but are not limited to greenfluorescent protein (GFP; from Aquorea and Renilla species), bluefluorescent protein (BFP), yellow fluorescent protein (YFP), and redfluorescent protein (RFP).

In a preferred embodiment, the reporter protein is Aequorea greenfluorescent protein or one of its variants; see Cody et al.,Biochemistry 32:1212-1218 (1993); and Inouye and Tsuji, FEBS Lett.341:277-280 (1994), both of which are expressly incorporated byreference herein. However, as is understood by those in the art,fluorescent proteins from other species may be used.

Accordingly, the present invention provides fusions of green fluorescentprotein (GFP) and random peptides. By “green fluorescent protein” or“GFP” herein is meant a protein with at least 30% sequence identity toGFP and exhibits fluorescence at 490 to 600 nm. The wild-type GFP is 238amino acids in length, contains a modified tripeptide fluorophore buriedinside a relatively rigid β-can structure which protects the fluorophorefrom the solvent, and thus solvent quenching. See Prasher et al., Gene111(2):229-233 (1992); Cody et al., Biochem. 32(5):1212-1218 (1993);Ormo et al, Science 273:1392-1395 (1996); and Yang et al., Nat. Biotech.14:1246-1251 (1996), all of which are hereby incorporated by referencein their entirety). Included within the definition of GFP arederivatives of GFP, including amino acid substitutions, insertions anddeletions. See for example WO 98/06737 and U.S. Pat. No. 5,777,079, bothof which are hereby incorporated by reference in their entirety.Accordingly, the GFP proteins utilized in the present invention may beshorter or longer than the wild type sequence. Thus, in a preferredembodiment, included within the definition of GFP proteins are portionsor fragments of the wild type sequence. For example, GFP deletionmutants can be made. At the N-terminus, it is known that only the firstamino acid of the protein may be deleted without loss of fluorescence.At the C-terminus, up to 7 residues can be deleted without loss offluorescence; see Phillips et al., Current Opin. Structural Biol. 7:821(1997).

For fusions involving fluorescent proteins other than GFP, proteins withat least 24% sequence homology to YFP, RFP, BFP are included with thescope of the present invention.

In a preferred embodiment, intein A is fused to the N-terminus of GFP.The fusion can be direct, i.e., with no additional residues between theC-terminus of intein A and the N-terminus of GFP, or indirect; that is,intervening amino acids are inserted between the N-terminus of GFP andthe C-terminus of intein A. See FIG. 7.

In a preferred embodiment, intein B is fused to the C-terminus of GFP.As above for N-terminal fusions, the fusion can be direct or indirect.

In a preferred embodiment, the reporter protein is an indirectlydetectable protein. As for the reporter proteins, cells that contain theindirectly detectable protein can be distinguished from those that donot; however, this is as a result of a secondary event. For example, apreferred embodiment utilizes “enzymatically detectable” reporters thatcomprise enzymes, such as luciferase, β-galactosidase, and β-lactamase,that will act on chromogenic and particularly fluorogenic substrates togenerate fluorescence. Alternatively, the indirectly detectable proteinmay require a recombinant construct in a cell that may be activated bythe reporter; for example, transcription factors or inducers that willbind to a promoter linked to an autofluorescent protein such thattranscription of the autofluorescent protein occurs.

In a preferred embodiment, the indirectly detectable protein is aDNA-binding protein which can bind to a DNA binding site and activatetranscription of an operably linked reporter gene. The reporter gene canbe any of the detectable genes, such as green fluorescent protein, orany of the survival genes outlined herein. The DNA binding site(s) towhich the DNA binding protein is binding is (are) placed proximal to abasal promoter that contains sequences required for recognition by thebasic transcription machinery (e.g., RNA polymerase II). The promotercontrols expression of a reporter gene. Following introduction of thischimeric reporter construct into an appropriate cell, an increase of thereporter gene product provides an indication that the DNA bindingprotein bound to its DNA binding site and activated transcription.Preferably, in the absence of the DNA binding protein, no reporter geneproduct is made. Alternatively, a low basal level of reporter geneproduct may be tolerated in the case when a strong increase in reportergene product is observed upon the addition of the DNA binding protein,or the DNA binding protein encoding gene. It is well known in the art togenerate vectors comprising DNA binding site(s) for a DNA bindingprotein to be analyzed, promoter sequences and reporter genes.

In a preferred embodiment, the DNA-binding protein is a cell typespecific DNA binding protein which can bind to a nucleic acid bindingsite within a promoter region to which endogenous proteins do not bindat all or bind very weakly. These cell type specific DNA-bindingproteins comprise transcriptional activators, such as Oct-2 (Mueller etal., Nature 336(6199):544-51 (1988)) which, e.g., is expressed inlymphoid cells and not in fibroblast cells. Expression of this DNAbinding protein in HeLa cells, which usually do not express thisprotein, is sufficient for a strong transcriptional activation of B-cellspecific promoters comprising a DNA binding site for Oct-2 (Mueller etal., supra).

In a preferred embodiment, the indirectly detectable protein is aDNA-binding/transcription activator fusion protein which can bind to aDNA binding site and activate transcription of an operably linkedreporter gene. Briefly, transcription can be activated through the useof two functional domains of a transcription activator protein; a domainor sequence of amino acids that recognizes and binds to a nucleic acidsequence, i.e., a nucleic acid binding domain, and a domain or sequenceof amino acids that will activate transcription when brought intoproximity to the target sequence. Thus the transcriptional activationdomain is thought to function by contacting other proteins required intranscription, essentially bringing in the machinery of transcription.It must be localized at the target gene by the nucleic acid bindingdomain, which putatively functions by positioning the transcriptionalactivation domain at the transcriptional complex of the target gene.

The DNA binding domain and the transcriptional activator domain can beeither from the same transcriptional activator protein, or can be fromdifferent proteins (see McKnight et al., Proc. Natl. Acad. Sci. USA89:7061 (1987); Ghosh et al., J. Mol. Biol. 234(3):610-619.(1993); andCurran et al., 55:395 (1988)). A variety of transcriptional activatorproteins comprising an activation domain and a DNA binding domain areknown in the art.

In a preferred embodiment, the DNA-binding/transcription activatorfusion protein is a tetracycline repressor protein (TetR)-VP16 fusionprotein. This bipartite fusion protein consists of a DNA binding domain(TetR) and a transcription activation domain (VP16). TetR binds withhigh specificity to the tetracycline operator sequence (tetO). The VP16domain is capable of activating gene expression of a gene of interest,provided that it is recruited to a functional promoter. Employing atetracycline repressor protein (TetR)-VP16 fusion protein, a suitableeukaryotic expression system which can be tightly controlled by theaddition or omission of tetracycline or doxycycline has been described(Gossen and Bujard, Proc. Natl. Acad. Sci. USA 89:5547-5551; Gossen etal., Science 268:1766-1769 (1995)).

It is an object of the instant application to fuse intein amino acidsequences to DNA-binding/transcription activator proteins and/or toDNA-binding/transcription activator fusion proteins. N-terminal andC-terminal fusions are all contemplated. The site of fusion may bedetermined based on the structure of DNA-binding/transcription activatorfusion protein, which are determined (e.g., TetR; see Orth et al., J.Mol. Biol. 285(2):455-61 (1999); Orth et al., J. Mol. Biol.279(2):439-47 (1998); Hinrichs et al., Science 264(5157):418-20(1994);and Kisker et al., J. Mol. Biol. 247(2):260-80 (1995)).

In a preferred embodiment, the reporter protein is a survival protein.By “survival protein”, “selection protein” or grammatical equivalentsherein is meant a protein without which the cell cannot survive, such asdrug resistance genes. As described herein, the cell usually does notnaturally contain an active form of the survival protein which is usedas a scaffold protein. As further described herein, the cell usuallycomprises a survival gene that encodes the survival protein.

The expression of a survival protein is usually not quantified in termsof protein activity, but rather recognized by conferring acharacteristic phenotype onto a cell which comprises the respectivesurvival gene or selection gene. Such survival genes may provideresistance to a selection agent (e.g., an antibiotic) to preferentiallyselect only those cells which contain and express the respectivesurvival gene. The variety of survival genes is quite broad andcontinues to grow (for review see Kriegler, Gene Transfer andExpression: A Laboratory Manual, W.H. Freeman and Company, New York,1990). Typically, the DNA containing the resistance-conferring phenotypeis transfected into a cell and subsequently the cell is treated withmedia containing the concentration of drug appropriate for the selectivesurvival and expansion of the transfected and now drug-resistant cells.

Selection agents such as ampicillin, kanamycin and tetracycline havebeen widely used for selection procedures in prokaryotes (e.g., seeWaxman and Strominger, Annu. Rev. Biochem. 52:825-69 (1983); Davies andSmith, Annu. Rev. Microbiol. 32:469-518 (1978); and Franklin, BiochemJ., 105(1):371-8 (1967)). Suitable selection agents for the selection ofeukaryotic cells include, but are not limited to, blasticidin (Izumi etal., Exp. Cell Res. 197(2):229-33 (1991); Kimura et al., Biochim.Biophys. Acta 1219(3):653-9 (1994); Kimura et al., Mol. Gen. Genet.242(2):121-9 (1994)), histidinol D (Hartman and Mulligan, Proc. Natl.Acad. Sci. USA. 85(21):8047-51 (1988)), hygromycin (Gritz and Davies,Gene 25(2-3):179-88 (1983); Sorensen et al., Gene 112(2):257-60 (1992)),neomycin (Davies and Jimenez, Am. J. Trop. Med. Hyg., 29(5Suppl):1089-92 (1980); Southern and Berg, J. Mol. Appl. Genet.1(4):327-41 (1982)), puromycin (de la Luna et al., Gene 62(1):121-6(1988)) and bleomycin/phleomycin/zeocin antibiotics (Mulsant et al.,Somat Cell. Mol. Genet. 14(3):243-52 (1988)).

Survival genes encoding enzymes mediating such a drug-resistantphenotype and protocols for their use are known in the art (seeKriegler, supra). Suitable survival genes include, but are not limitedto, thymidine kinase (TK; Wigler et al., Cell 11:233 (1977)), adeninephosphoribosyltransferase (APRT; Lowry et al., Cell 22:817 (1980);Murray et al., Gene 31:233 (1984); Stambrook et al., Som. Cell. Mol.Genet. 4:359 (1982)), hypoxanthine-guanine phosphoribosyltransferase(HGPRT; Jolly et al., Proc. Natl. Acad. Sci. USA 80:477 (1983)),dihydrofolate reductase (DHFR; Subramani et al., Mol. Cell. Biol. 1:854(1985); Kaufman and Sharp, J. Mol. Biol. 159:601 (1982); Simonsen andLevinson, Proc. Natl. Acad. Sci. USA. 80:2495 (1983)), aspartatetranscarbamylase (Ruiz and Wahl, Mol. Cell. Biol. 6:3050 (1986)),ornithine decarboxylase (Chiang and McConlogue, Mol. Cell. Biol. 8:764(1988)), aminoglycoside phosphotrarisferase (Southern and Berg, Mol.Appl. Gen. 1:327 (1982); Davies and Jiminez, supra)),hygromycin-B-phosphotransferase (Gritz and Davies, supra; Sugden et al.,Mol. Cell. Biol. 5:410 (1985); Palmer et al., Proc. Natl. Acad. Sci. USA84:1055 (1987)), xanthine-guanine phosphoribosyltransferase (Mulliganand Berg, Proc. Natl. Acad. Sci. USA 78:2072 (1981)), tryptophansynthetase (Hartman and Mulligan, Proc. Natl. Acad. Sci. USA 85:8047(1988)), histidinol dehydrogenase (Hartman and Mulligan, supra),multiple drug resistance biochemical marker (Kane et al., Mol. Cell.Biol. 8:3316 (1988); Choi et al., Cell 53:519 (1988)), blasticidin Sdeaminase (Izumi et al., Exp. Cell. Res. 197(2):229-33 (1991)),bleomycin hydrolase (Mulsant et al., supra), andpuromycin-N-acetyl-transferase (Lacalle et al., Gene 79(2):375-80(1989)),

In another preferred embodiment, the survival protein is blasticidin Sdeaminase, which is encoded by the bsr gene (Izumi et al., Exp. Cell.Res. 197(2):229-33 (1991)). When transferred into almost any cell, thisdominant selectable gene confers resistance to media comprising theantibiotic blasticidin S. Blasticidin S deaminase encoding genes havebeen cloned. They are used widely as a selectable marker on variousvectors and the nucleotide sequences are available (e.g., see GenBankaccession numbers D83710, U75992, and U75991).

It is an object of the instant application to fuse intein motifsequences to blasticidin S deaminase. N-terminal and C-terminal fusionsare all contemplated. The site of fusion may be determined based on thestructure of Aspergillus terreus blasticidin S deaminase, which has beendetermined (Nakasako et al., Acta Crystallogr. D. Biol. Crystallogr.55(Pt2):547-8 (1999)). Also, internal fusions can be done; see PCTUS99/23715, hereby incorporated by reference.

In another preferred embodiment, the survival protein ispuromycin-N-acetyl-transferase, which is encoded by the pac gene(Lacalle et al., Gene 79(2):375-80 (1989)). When transferred into almostany cell, this dominant selectable gene confers resistance to mediacomprising puromycin. A puromycin-N-acetyltransferase encoding gene hasbeen cloned. It is used widely as a selectable marker on various vectorsand the nucleotide sequences are available (e.g., see GenBank accessionnumbers Z75185 and M25346).

It is an object of the instant application to fuse intein motifsequences puromycin-N-acetyl-transferase. N-terminal and C-terminal,dual N— and C-terminal and one or more internal fusions are allcontemplated.

In a preferred embodiment, in addition to the intein motifs andpeptides, the fusion polypeptides of the present invention preferablyinclude additional components, including, but not limited to, fusionpartners.

By “fusion partner” herein is meant a sequence that is associated withthe fusion polypeptide that confers upon all members of the library inthat class a common function or ability. Fusion partners can beheterologous (i.e., not native to the host cell), or synthetic (i.e.,not native to any cell). Suitable fusion partners include, but are notlimited to: a) targeting sequences, defined below, which allow thelocalization of the peptide into a subcellular or extracellularcompartment; b) rescue sequences as defined below, which allow thepurification or isolation of either the peptides or the nucleic acidsencoding them; or c), any combination of a) and b).

In a preferred embodiment, the fusion partner is a targeting sequence.As will be appreciated by those in the art, the localization of proteinswithin a cell is a simple method for increasing effective concentrationand determining function. For example, RAF1 when localized to themitochondrial membrane can inhibit the anti-apoptotic effect of BCL-2.Similarly, membrane bound Sos induces Ras mediated signaling inT-lymphocytes. These mechanisms are thought to rely on the principle oflimiting the search space for ligands, that is to say, the localizationof a protein to the plasma membrane limits the search for its ligand tothat limited dimensional space near the membrane as opposed to the threedimensional space of the cytoplasm. Alternatively, the concentration ofa protein can also be simply increased by nature of the localization.Shuttling the proteins into the nucleus confines them to a smaller spacethereby increasing concentration. Finally, the ligand or target maysimply be localized to a specific compartment, and inhibitors must belocalized appropriately.

Thus, suitable targeting sequences include, but are not limited to,binding sequences capable of causing binding of the expression productto a predetermined molecule or class of molecules while retainingbioactivity of the expression product, (for example by using enzymeinhibitor or substrate sequences to target a class of relevant enzymes);sequences signalling selective degradation, of itself or co-boundproteins; and signal sequences capable of constitutively localizing thepeptides to a predetermined cellular locale, including a) subcellularlocations such as the Golgi, endoplasmic reticulum, nucleus, nucleoli,nuclear membrane, mitochondria, chloroplast, secretory vesicles,lysosome, and cellular membrane; and b) extracellular locations via asecretory signal. Particularly preferred is localization to eithersubcellular locations or to the outside of the cell via secretion. SeeFIG. 8.

In a preferred embodiment, the targeting sequence is a nuclearlocalization signal (NLS). NLSs are generally short, positively charged(basic) domains that serve to direct the entire protein in which theyoccur to the cell's nucleus. Numerous NLS amino acid sequences have beenreported including single basic NLS's such as that of the SV40 (monkeyvirus) large T Antigen (Pro Lys Lys Lys Arg Lys Val), Kalderon et al.,Cell, 39:499-509 (1984)); the human retinoic acid receptor-β nuclearlocalization signal (ARRRRP); NF—KB p50 (EEVQRKRQKL; Ghosh et al., Cell62:1019 (1990)); NF—KB p65 (EEKRKRTYE; Nolan et al., Cell 64:961(1991)); and others (see for example Boulikas, J. Cell. Biochem.55(1):32-58 (1994), hereby incorporated by reference) and double basicNLS's exemplified by that of the Xenopus (African clawed toad) protein,nucleoplasmin (Ala Val Lys Arg Pro Ala Ala Thr Lys Lys Ala Gly Gln AlaLys Lys Lys Lys Leu Asp), Dingwall, et al., Cell, 30:449-458, 1982 andDingwall, et al., J. Cell Biol., 107:641-849; 1988)). Numerouslocalization studies have demonstrated that NLSs incorporated insynthetic peptides or grafted onto reporter proteins not normallytargeted to the cell nucleus cause these peptides and reporter proteinsto be concentrated in the nucleus. See, for example, Dingwall, andLaskey, Ann, Rev. Cell Biol., 2:367-390 (1986); Bonnerot et al., Proc.Natl. Acad. Sci. USA, 84:6795-6799 (1987); and Galileo et al., Proc.Natl. Acad. Sci. USA, 87:458-462 (1990).

In a preferred embodiment, the targeting sequence is a membraneanchoring signal sequence. This is particularly useful since manyparasites and pathogens bind to the membrane, in addition to the factthat many intracellular events originate at the plasma membrane. Thus,membrane-bound peptide libraries are useful for both the identificationof important elements in these processes as well as for the discovery ofeffective inhibitors. The invention provides methods for presenting therandomized expression product extracelluarly or in the cytoplasmicspace. For extracellular presentation, a membrane anchoring region isprovided at the carboxyl terminus of the peptide presentation structure.The randomized expression product region is expressed on the cellsurface and presented to the extracellular space, such that it can bindto other surface molecules (affecting their function) or moleculespresent in the extracellular medium. The binding of such molecules couldconfer function on the cells expressing a peptide that binds themolecule. The cytoplasmic region could be neutral or could contain adomain that, when the extracellular randomized expression product regionis bound, confers a function on the cells (activation of a kinase,phosphatase, binding of other cellular components to effect function).Similarly, the randomized expression product-containing region could becontained within a cytoplasmic region, and the transmembrane region andextracellular region remain constant or have a defined function.

Membrane-anchoring sequences are well known in the art and are based onthe genetic geometry of mammalian transmembrane molecules. Peptides areinserted into the membrane based on a signal sequence (designated hereinas ssTM) and require a hydrophobic transmembrane domain (herein TM). Thetransmembrane proteins are inserted into the membrane such that theregions encoded 5′ of the transmembrane domain are extracellular and thesequences 3′ become intracellular. Of course, if these transmembranedomains are placed 5′ of the variable region, they will serve to anchorit as an intracellular domain, which may be desirable in someembodiments. ssTMs and TMs are known for a wide variety of membranebound proteins, and these sequences may be used accordingly, either aspairs from a particular protein or with each component being taken froma different protein, or alternatively, the sequences may be synthetic,and derived entirely from consensus as artificial delivery domains.

As will be appreciated by those in the art, membrane-anchoringsequences, including both ssTM and TM, are known for a wide variety ofproteins and any of these may be used. Particularly preferredmembrane-anchoring sequences include, but are not limited to, thosederived from CD8, ICAM-2, IL-8R, CD4 and LFA-1.

Useful sequences include sequences from: 1) class I integral membraneproteins such as IL-2 receptor β-chain (residues 1-26 are the signalsequence, 241-265 are the transmembrane residues; see Hatakeyama et al.,Science 244:551 (1989) and von Heijne et al, Eur. J. Biochem. 174:671(1988)) and insulin receptor β-chain (residues 1-27 are the signal,957-959 are the transmembrane domain and 960-1382 are the cytoplasmicdomain; see Hatakeyama, supra, and Ebina et al., Cell 40:747 (1985)); 2)class II integral membrane proteins such as neutral endopeptidase(residues 29-51 are the transmembrane domain, 2-28 are the cytoplasmicdomain; see Malfroy et al., Biochem. Biophys. Res. Commun. 144:59(1987)); 3) type III proteins such as human cytochrome P450 NF25(Hatakeyama, supra); and 4) type IV proteins such as humanP-glycoprotein (Hatakeyama, supra). Particularly preferred are CD8 andICAM-2. For example, the signal sequences from CD8 and ICAM-2 lie at theextreme 5′ end of the transcript. These consist of the amino acids 1-32in the case of CD8 (MASPLTRFLSLNLLLLGESILGSGEAKPQAP; Nakauchi et al.,Proc. Natl. Acad. Sci. USA 82:5126 (1985)) and 1-21 in the case ofICAM-2 (MSSFGYRTLTVALFTLICCPG; Staunton et al., Nature 339:61 (1989)).These leader sequences deliver the construct to the membrane while thehydrophobic transmembrane domains, placed 3′ of the random peptideregion, serve to anchor the construct in the membrane. Thesetransmembrane domains are encompassed by amino acids 145-195 from CD8(PQRPEDCRPRGSVKGTGLDFACDIYIWAPLAGICVALLLSLIITLICYHSR; Nakauchi, supra)and 224-256 from ICAM-2 (MVIIVTVVSVLLSLFVTSVLLCFIFGQHLRQQR; Staunton,supra).

Alternatively, membrane anchoring sequences include the GPI anchor,which results in a covalent bond between the molecule and the lipidbilayer via a glycosyl-phosphatidylinositol bond for example in DAF(PNKGSGTTSGTTRLLSGHTCFTLTGLLGTLVTMGLLT, with the bolded serine the siteof the anchor; see Homans et al., Nature 333(6170):269-72 (1988), andMoran et al., J. Biol. Chem. 266:1250 (1991)). In order to do this, theGPI sequence from Thy-1 can be cassetted 3′ of the variable region inplace of a transmembrane sequence.

Similarly, myristylation sequences can serve as membrane anchoringsequences. It is known that the myristylation of c-src recruits it tothe plasma membrane. This is a simple and effective method of membranelocalization, given that the first 14 amino acids of the protein aresolely responsible for this function: MGSSKSKPKDPSQR (see Cross et al.,Mol. Cell. Biol. 4(9):1834 (1984); Spencer et al., Science 262:1019-1024(1993), both of which are hereby incorporated by reference). This motifhas already been shown to be effective in the localization of reportergenes and can be used to anchor the zeta chain of the TCR. This motif isplaced 5′ of the variable region in order to localize the construct tothe plasma membrane. Other modifications such as palmitoylation can beused to anchor constructs in the plasma membrane; for example,palmitoylation sequences from the G protein-coupled receptor kinase GRK6sequence (LLQRLFSRQDCCGNCSDSEEELPTRL, with the bold cysteines beingpalmitolyated; Stoffel et al., J. Biol. Chem 269:27791 (1994)); fromrhodopsin (KQFRNCMLTSLCCGKNPLGD; Barnstable et al., J. Mol. Neurosci.5(3):207 (1994)); and the p21 H-ras 1 protein (LNPPDESGPGCMSCKCVLS;Capon et al., Nature 302:33 (1983)).

In a preferred embodiment, the targeting sequence is a lysozomaltargeting sequence, including, for example, a lysosomal degradationsequence such as Lamp-2 (KFERQ; Dice, Ann. N.Y. Acad. Sci. 674:58(1992)); or lysosomal membrane sequences from Lamp-1(MLIPIAGFFALAGLVLIVLIAYLIGRKRSHAGYQTI, Uthayakumar et al., Cell. Mol.Biol. Res. 41:405 (1995)) or Lamp-2(LVPIAVGAALAGVLILVLLAYFIGLKHHHAGYEQF, Konecki et la., Biochem. Biophys.Res. Comm. 205:1-5 (1994)), both of which show the transmembrane domainsin italics and the cytoplasmic targeting signal underlined).

Alternatively, the targeting sequence may be a mitrochondriallocalization sequence, including mitochondrial matrix sequences (e.g.,yeast alcohol dehydrogenase III; MLRTSSLFTRRVQPSLFSRNILRLQST; Schatz,Eur. J. Biochem. 165:1-6 (1987)); mitochondrial inner membrane sequences(yeast cytochrome c oxidase subunit IV; MLSLRQSIRFFKPATRTLCSSRYLL;Schatz, supra); mitochondrial intermembrane space sequences (yeastcytochrome c1;MFSMLSKRWAQRTLSKSFYSTATGMSKSGKLTQKLVTAGVAAAGITASTLLYADSLTAEAMTA; Schatz,supra) or mitochondrial outer membrane sequences (yeast 70 kD outermembrane protein; MKSFITRNKTAILATVMTGTAIGAYYYYNQLQQQQQRGKK; Schatz,supra).

The target sequences may also be endoplasmic reticulum sequences,including the sequences from calreticulin (KDEL; Pelham, Royal SocietyLondon Transactions B; 1-10 (1992)) or adenovirus E3/19K protein(LYLSRRSFIDEKKMP; Jackson et al., EMBO J. 9:3153 (1990)).

Furthermore, targeting sequences also include peroxisome sequences (forexample, the peroxisome matrix sequence from Luciferase; SKL; Keller etal., Proc. Natl. Acad. Sci. USA 4:3264 (1987)); farnesylation sequences(for example, P21 H-ras 1; LNPPDESGPGCMSCKCVLS, with the bold cysteinefarnesylated; Capon, supra); geranylgeranylation sequences (for example,protein rab-5A; LTEPTQPTRNQCCSN, with the bold cysteinesgeranylgeranylated; Farnsworth, Proc. Natl. Acad. Sci. USA 91:11963(1994)); or destruction sequences (cyclin B1; RTALGDIGN; Klotzbucher etal., EMBO J. 1:3053 (1996)).

In a preferred embodiment, the targeting sequence is a secretory signalsequence capable of effecting the secretion of the peptide. There are alarge number of known secretory signal sequences which are placed 5′ tothe variable peptide region, and are cleaved from the peptide region toeffect secretion into the extracellular space. Secretory signalsequences and their transferability to unrelated proteins are wellknown, e.g., Silhavy, et al. Microbiol. Rev. 49, 398-418 (1985)). Thisis particularly useful to generate a peptide capable of binding to thesurface of, or affecting the physiology of, a target cell that is otherthan the host cell, e.g., the cell infected with the retrovirus. In apreferred approach, a fusion polypeptide is configured to contain, inseries, a secretion signal peptide-intein B motif-randomized librarysequence-intein A. See FIG. 8. In this manner, target cells grown in thevicinity of cells caused to express the library of peptides, are bathedin secreted peptide. Target cells exhibiting a physiological change inresponse to the presence of a peptide, e.g., by the peptide binding to asurface receptor or by being internalized and binding to intracellulartargets, and the secreting cells are localized by any of a variety ofselection schemes and the peptide causing the effect determined.Exemplary effects include variously that of a designer cytokine (i.e., astem cell factor capable of causing hematopoietic stem cells to divideand maintain their totipotential), a factor causing cancer cells toundergo spontaneous apoptosis, a factor that binds to the cell surfaceof target cells and labels them specifically, etc.

Suitable secretory sequences are known, including signals from IL-2(MYRMQLLSCIALSLALVTNS; Villinger et al., J. Immunol. 155:3946 (1995)),growth hormone (MATGSRTSLLLAFGLLCLPWLQEGSAFPT; Roskam et al., NucleicAcids Res. 7:30 (1979)); preproinsulin (MALWMRLLPLLALLALWGPDPAAAFVN;Bell et al., Nature 284:26 (1980)); and influenza HA protein(MKAKLLVLLYAFVAGDQI; Sekiwawa et al., Proc. Natl. Acad. Sci. USA80:3563)), with cleavage between the non-underlined-underlined junction.A particularly preferred secretory signal sequence is the signal leadersequence from the secreted cytokine IL-4, which comprises the first 24amino acids of IL-4 as follows: MGLTSQLLPPLFFLLACAGNFVHG.

In a preferred embodiment, the fusion partner is a rescue sequence. Arescue sequence is a sequence which may be used to purify or isolateeither the peptide or the nucleic acid encoding it. Thus, for example,peptide rescue sequences include purification sequences such as the His₆tag for use with Ni affinity columns and epitope tags for detection,immunoprecipitation or FACS (fluorescence-activated cell sorting).Suitable epitope tags include myc (for use with the commerciallyavailable 9E10 antibody), the BSP biotinylation target sequence of thebacterial enzyme BirA, flu tags, lacZ, GST, and Strep tag I and II.

Alternatively, the rescue sequence may be a unique oligonucleotidesequence which serves as a probe target site to allow the quick and easyisolation of the retroviral construct, via PCR, related techniques, orhybridization.

While the discussion has been directed to the fusion of fusion partnersto the intein portion of the fusion polypeptide, the fusion partners maybe placed anywhere (i.e. N-terminal, C-terminal, internal) in thestructure as the biology and activity permits. In addition, it is alsopossible to fuse one or more of these fusion partners to the inteinportions of the fusion polypeptide. Thus, for example, a targetingsequence (either N-terminally, C-terminally, or internally, as describedbelow) may be fused to intein A, and a rescue sequence fused to the sameplace or a different place on the molecule. Thus, any combination offusion partners and peptides may be made.

In a preferred embodiment, the invention provides libraries of fusionpolypeptides. By “library” herein is meant a sufficiently structurallydiverse population of randomized expression products to effect aprobabilistically sufficient range of cellular responses to provide oneor more cells exhibiting a desired response. Accordingly, an interactionlibrary must be large enough so that at least one of its members willhave a structure that gives it affinity for some molecule, protein, orother factor whose activity is of interest. Although it is difficult togauge the required absolute size of an interaction library, natureprovides a hint with the immune response: a diversity of 10⁷-10⁸different antibodies provides at least one combination with sufficientaffinity to interact with most potential antigens faced by an organism.Published in vitro selection techniques have also shown that a librarysize of 10⁷ to 10⁸ is sufficient to find structures with affinity forthe target. A library of all combinations of a peptide 7 to 20 aminoacids in length, such as proposed here for expression in retroviruses,has the potential to code for 20⁷ (10⁹) to 20²⁰. Thus, with libraries of10⁷ to 10⁸ per ml of retroviral particles the present methods allow a“working” subset of a theoretically complete interaction library for 7amino acids, and a subset of shapes for the 20²⁰ library. Thus, in apreferred embodiment, at least 10⁶, preferably at least 10⁷ morepreferably at least 10⁸ and most preferably at least 10⁹ differentexpression products are simultaneously analyzed in the subject methods.Preferred methods maximize library size and diversity.

In a preferred embodiment, libraries of all combinations of a peptide 3to 30 amino acids in length are synthesized and analyzed as outlinedherein. Libraries of smaller cyclic peptides, i.e., 3 to 4 amino acid inlength, are advantageous because they are more constrained and thusthere is a better chance that these libraries possess desirablepharmocokinetics properties as a consequence of their smaller size.Accordingly, the libraries of the present invention may be one of any ofthe following lengths: 3 amino acids, 4 amino acids, 5 amino acids, 6amino acids, 7 amino acids, 8 amino acids, 9 amino acids, 10 aminoacids, 11 amino acids, 12 amino acids, 13 amino acids, 14 amino acids,15 amino acids, 16 amino acids, 17 amino acids, 18 amino acids, 19 aminoacids, 20 amino acids, 21 amino acids, 22 amino acids, 23 amino acids,24 amino acids, 25 amino acids, 26 amino acids, 27 amino acids, 28 aminoacids, 29 amino acids and 30 amino acids in length.

That is, in some embodiments the library is random, i.e., allcombinations of a peptide of a given length are encoded. However, inother alternative embodiments, the library is biased as set forth below.

The invention further provides fusion nucleic acids encoding the fusionpolypeptides of the invention. As will be appreciated by those in theart, due to the degeneracy of the genetic code, an extremely largenumber of nucleic acids may be made, all of which encode the fusionproteins of the present invention. Thus, having identified a particularamino acid sequence, those skilled in the art could make any number ofdifferent nucleic acids, by simply modifying the sequence of one or morecodons in a way which does not change the amino acid sequence of thefusion protein.

Using the nucleic acids of the present invention which encode a fusionprotein, a variety of expression vectors are made. The expressionvectors may be either self-replicating extrachromosomal vectors orvectors which integrate into a host genome. Generally, these expressionvectors include transcriptional and translational regulatory nucleicacid operably linked to the nucleic acid encoding the fusion protein.The term “control sequences” refers to DNA sequences necessary for theexpression of an operably linked coding sequence in a particular hostorganism. The control sequences that are suitable for prokaryotes, forexample, include a promoter, optionally an operator sequence, and aribosome binding site. Eukaryotic cells are known to utilize promoters,polyadenylation signals, and enhancers.

The fusion nucleic acids are introduced into cells to screen for cyclicpeptides capable of altering the phenotype of a cell. By “introducedinto ” or grammatical equivalents herein is meant that the nucleic acidsenter the cells in a manner suitable for subsequent expression of thenucleic acid. The method of introduction is largely dictated by thetargeted cell type, discussed below. Exemplary methods include CaPO₄precipitation, liposome fusion, lipofectin®, electroporation, viralinfection, etc. The fusion nucleic acids may stably integrate into thegenome of the host cell (for example, with retroviral introduction,outlined below), or may exist either transiently or stably in thecytoplasm (e.g., through the use of traditional plasmids, utilizingstandard regulatory sequences, selection markers, etc.). As manypharmaceutically important screens require human or model mammalian celltargets, retroviral vectors capable of transfecting such targets arepreferred.

In a preferred embodiment, the fusion nucleic acids are part of aretroviral particle which infects the cells. Generally, infection of thecells is straightforward with the application of the infection-enhancingreagent polybrene, which is a polycation that facilitates viral bindingto the target cell. Infection can be optimized such that each cellgenerally expresses a single construct, using the ratio of virusparticles to number of cells. Infection follows a Poisson distribution.

In a preferred embodiment, the fusion nucleic acids are introduced intocells using retroviral vectors. Currently, the most efficient genetransfer methodologies harness the capacity of engineered viruses, suchas retroviruses, to bypass natural cellular barriers to exogenousnucleic acid uptake. The use of recombinant retroviruses was pioneeredby Richard Mulligan and David Baltimore with the Psi-2 lines andanalogous retrovirus packaging systems, based on NIH 3T3 cells (see Mannet al., Cell 33:153-159 (1993), hereby incorporated by reference). Suchhelper-defective packaging lines are capable of producing all thenecessary trans proteins -gag, pol, and env- that are required forpackaging, processing, reverse transcription, and integration ofrecombinant genomes. Those RNA molecules that have in cis the Kpackaging signal are packaged into maturing virions. Retroviruses arepreferred for a number of reasons. First, their derivation is easy.Second, unlike Adenovirus-mediated gene delivery, expression fromretroviruses is long-term (adenoviruses do not integrate).Adeno-associated viruses have limited space for genes and regulatoryunits and there is some controversy as to their ability to integrate.Retroviruses therefore offer the best current compromise in terms oflong-term expression, genomic flexibility, and stable integration, amongother features. The main advantage of retroviruses is that theirintegration into the host genome allows for their stable transmissionthrough cell division. This ensures that in cell types which undergomultiple independent maturation steps, such as hematopoietic cellprogression, the retrovirus construct will remain resident and continueto express.

A particularly well suited retroviral transfection system is describedin Mann et al., supra: Pear et al., Proc. Natl. Acad. Sci. USA90(18):8392-6 (1993); Kitamura et al., Proc. Natl. Acad. Sci. USA92:9146-9150 (1995); Kinsella et al., Human Gene Therapy 7:1405-1413(1996); Hofmann et al., Proc. Natl. Acad. Sci. USA 93:5185-5190 (1996);Choate et al., Human Gene Therapy 7:2247 (1996); and WO 94/19478; andreferences cited therein, all of which are incorporated by reference.

In one embodiment of the invention, the library is generated in aintein-catalyzed cyclization scaffold. By “intein-catalyzed cyclizationscaffold” herein is meant that the intein is engineered such that acyclic peptide is generated upon intein-mediated splicing of theextein-intein junction points. Preferably, an intein cyclizationscaffold includes the C-terminal intein motif, a library insert of 3 upto 30 amino acids in length, and the N-terminal intein motif. The C— andN-terminal intein motifs can be derived from any number of known inteinscapable mediating protein splicing, including split-inteins. Mostwild-type inteins have requirements for a specific extein-encoded aminoacid at the C-intein (IntB)/C-extein junction point. This variesdepending on the intein, but most often consists of an cysteine,threonine or serine. Intein-generated cyclic peptide libraries may begenerated in which this particular amino acid is fixed and correspondsto the amino acid present in the wild-type sequence. For example, theSsp. DnaB intein utilizes an extein-encoded serine in this position.

A number of inteins have the ability to catalyze protein splicing whennon-native amino acids are substituted at the C-intein (IntB)/C-exteinjunction point position. Degeneracy at the C-intein (IntB)/C-exteinjunction point position leads to cyclic peptide libraries of greatercomplexity and therefore added utility. The proposed degeneracy in thisposition most likely consists of a cysteine, serine or threonine but isnot limited to these amino acids. The ability of a givenintein-catalyzed cyclization scaffold to tolerate degeneracy at thisposition depends on the specific intein utilized and it's mechanism ofprotein splicing. Thus, isolation of intein cyclization scaffolds with agreater tolerance for degeneracy at the C-intein (IntB)/C-exteinjunction point is within the scope of this invention.

In one embodiment of the invention, the library is generated in aretrovirus DNA construct backbone, as is generally described in U.S.Ser. No. 08/789,333, filed Jan. 23, 1997, incorporated herein byreference. Standard oligonucleotide synthesis is done to generate therandom portion of the candidate bioactive agent, using techniques wellknown in the art (see Eckstein, Oligonucleotides and Analogues, APractical Approach, IRL Press at Oxford University Press, 1991);libraries may be commercially purchased. Libraries with up to 10⁹ to10¹⁰ unique sequences can be readily generated in such DNA backbones.After generation of the DNA library, the library is cloned into a firstprimer. The first primer serves as a “cassette”, which is inserted intothe retroviral construct. The first primer generally contains a numberof elements, including for example, the required regulatory sequences(e.g., translation, transcription, promoters, etc), fusion partners,restriction endonuclease (cloning and subcloning) sites, stop codons(preferably in all three frames), regions of complementarity for secondstrand priming (preferably at the end of the stop codon region as minordeletions or insertions may occur in the random region), etc. See U.S.Ser. No. 08/789,333, hereby incorporated by reference.

A second primer is then added, which generally consists of some or allof the complementarity region to prime the first primer and optionalnecessary sequences for a second unique restriction site for subcloning.DNA polymerase is added to make double-stranded oligonucleotides. Thedouble-stranded oligonucleotides are cleaved with the appropriatesubcloning restriction endonucleases and subcloned into the targetretroviral vectors, described below.

Any number of suitable retroviral vectors may be used. Generally, theretroviral vectors may include: selectable marker genes under thecontrol of internal ribosome entry sites (IRES), which allows forbicistronic operons and thus greatly facilitates the selection of cellsexpressing peptides at uniformly high levels; and promoters drivingexpression of a second gene, placed in sense or anti-sense relative tothe 5′ LTR. Suitable selection genes include, but are not limited to,neomycin, blastocidin, bleomycin, puromycin, and hygromycin resistancegenes, as well as self-fluorescent markers such as green fluorescentprotein, enzymatic markers such as lacZ, and surface proteins such asCD8, etc.

Preferred vectors include a vector based on the murine stem cell virus(MSCV) (see Hawley et al., Gene Therapy 1:136 (1994)) and a modified MFGvirus (Rivere et al., Genetics 92:6733 (1995)), and pBABE, outlined inthe examples. A general schematic of the retroviral construct isdepicted in FIGS. 6 and 15.

The retroviruses may include inducible and constitutive promoters. Forexample, there are situations wherein it is necessary to induce peptideexpression only during certain phases of the selection process. Forinstance, a scheme to provide pro-inflammatory cytokines in certaininstances must include induced expression of the peptides. This isbecause there is some expectation that over-expressed pro-inflammatorydrugs might in the long-term be detrimental to cell growth. Accordingly,constitutive expression is undesirable, and the peptide is only turnedon during that phase of the selection process when the phenotype isrequired, and then shut the peptide down by turning off the retroviralexpression to confirm the effect or ensure long-term survival of theproducer cells. A large number of both inducible and constitutivepromoters are known.

In addition, it is possible to configure a retroviral vector to allowinducible expression of retroviral inserts after integration of a singlevector in target cells; importantly, the entire system is containedwithin the single retrovirus. Tet-inducible retroviruses have beendesigned incorporating the Self-Inactivating (SIN) feature of 3′ LTRenhancer/promoter retroviral deletion mutant (Hoffman et al., Proc.Natl. Acad. Sci USA USA 93:5185 (1996)). Expression of this vector incells is virtually undetectable in the presence of tetracycline or otheractive analogs. However, in the absence of Tet, expression is turned onto maximum within 48 hours after induction, with uniform increasedexpression of the whole population of cells that harbor the inducibleretrovirus, indicating that expression is regulated uniformly within theinfected cell population. A similar, related system uses a mutated TetDNA-binding domain such that it bound DNA in the presence of Tet, andwas removed in the absence of Tet. Either of these systems is suitable.

In this manner the primers create a library of fragments, eachcontaining a different random nucleotide sequence that may encode adifferent peptide. The ligation products are then transformed intobacteria, such as E. coli, and DNA is prepared from the resultinglibrary, as is generally outlined in Kitamura, Proc. Natl. Acad. Sci.USA 92:9146-9150 (1995), hereby expressly incorporated by reference.

Delivery of the library DNA into a retroviral packaging system resultsin conversion to infectious virus. Suitable retroviral packaging systemcell lines include, but are not limited to, the Bing and BOSC23 celllines described in WO 94/19478; Soneoka et al., Nucleic Acid Res.23(4):628 (1995); Finer et al., Blood 83:43 (1994); Pheonix packaginglines such as PhiNX-eco and PhiNX-ampho, described below; 292T+gag-poland retrovirus envelope; PA317; and cell lines outlined in Markowitz etal., Virology 167:400 (1988), Markowitz et al., J. Virol. 62:1120(1988), Li et al., Proc. Natl. Acad. Sci. USA 93:11658 (1996), Kinsellaet al., Human Gene Therapy 7:1405 (1996), all of which are incorporatedby reference.

Preferred systems include PHEONIX-ECO and PHEONIX-AMPHO. BothPHEONIX-ECO and PHEONIX-AMPHO were tested for helper virus productionand established as being helper-virus free. Both lines can carryepisomes for the creation of stable cell lines which can be used toproduce retrovirus. Both lines are readily testable by flow cytometryfor stability of gag-pol (CD8) and envelope expression; after severalmonths of testing the lines appear stable, and do not demonstrate lossof titre as did the first-generation lines BOSC23 and Bing (partly dueto the choice of promoters driving expression of gag-pol and envelope).Both lines can also be used to transiently produce virus in a few days.Thus, these lines are fully compatible with transient, episomal stable,and library generation for retroviral gene transfer experiments.Finally, the titres produced by these lines have been tested. Usingstandard polybrene-enhanced retroviral infection, titres approaching orabove 10⁷ per ml were observed for both PHEONIX-eco and PHEONIX-amphowhen carrying episomal constructs. When transiently produced virus ismade, titres are usually ½ to ⅓ that value.

These lines are helper-virus free, carry episomes for long-term stableproduction of retrovirus, stably produce gag-pol and env, and do notdemonstrate loss of viral titre over time. In addition, PhiNX-eco andPhiNX-ampho are capable of producing titres approaching or above 10⁷ perml when carrying episomal constructs, which, with concentration ofvirus, can be enhanced to 10⁸ to 10⁹ per ml.

In a preferred embodiment, the cell lines disclosed above, and the othermethods for producing retrovirus, are useful for production of virus bytransient transfection. The virus can either be used directly or be usedto infect another retroviral producer cell line for “expansion” of thelibrary.

Concentration of virus may be done as follows. Generally, retrovirusesare titred by applying retrovirus-containing supernatant onto indicatorcells, such as NIH3T3 cells, and then measuring the percentage of cellsexpressing phenotypic consequences of infection. The concentration ofthe virus is determined by multiplying the percentage of cells infectedby the dilution factor involved, and taking into account the number oftarget cells available to obtain a relative titre. If the retroviruscontains a reporter gene, such as lacZ, then infection, integration, andexpression of the recombinant virus is measured by histological stainingfor lacZ expression or by flow cytometry (FACS). In general, retroviraltitres generated from even the best of the producer cells do not exceed10⁷ per ml, unless concentration by relatively expensive or exoticapparatus. However, as it has been recently postulated that since aparticle as large as a retrovirus will not move very far by brownianmotion in liquid, fluid dynamics predicts that much of the virus nevercomes in contact with the cells to initiate the infection process.However, if cells are grown or placed on a porous filter and retrovirusis allowed to move past cells by gradual gravitometric flow, a highconcentration of virus around cells can be effectively maintained at alltimes. Thus, up to a ten-fold higher infectivity by infecting cells on aporous membrane and allowing retrovirus supernatant to flow past themhas been seen. This should allow titres of 10⁹ after concentration.

The fusion nucleic acids and polypeptides of the invention are used tomake cyclic peptides. By “cyclic peptides” or grammatical equivalentsherein is meant the intracellular catalysis of peptide backbonecyclization. Generally, backbone cyclization results in the joining ofthe N and C termini of a peptide together such that a cyclic product isgenerated inside cells.

Preferably, every member of a peptide library is tested for bioactivityusing one of the assays described below. For example, a cyclic peptidewith 7 random positions has a complexity of 20⁷=1.28×10⁹, all of whichwill be tested for biological activity.

In the event it is not possible to test every member of a library forbioactivity, the library may be deliberately biased. For example, acyclic peptide can be biased to cellular entry by fixing one or morerelatively hydrophobic amino acids, such as tyrosine or tryptophan.Other types of biased libraries which may be synthesized includelibraries which primary contain cyclic peptides comprising amino acidswith large side chains and libraries in which the number of cyclicpeptide conformers is restricted.

In addition, the library may be biased to encode or include peptidesthat target particular proteins. For example, when two proteins areknown to interact, a cyclic peptide library may be used that targets theknown protein-protein interaction site. The library members may includethe sequence of the interaction sites of the proteins. That is, thesequence of the interacting proteins at the site of interaction may beincluded within the library. In this system, the library member maycompete with the interacting molecules for binding. However, inhibitionneed not be a result of competitive inhibition. That is, inhibition ofprotein-protein interaction may be accomplished by a variety ofmechanisms including competitive inhibition, non-competitive inhibition,and un-competitive inhibition. Protein-protein interaction may beinhibited as a result of allosteric mechanisms. That is, library memberbinding to a site on a protein that is different from theprotein-protein interaction site may result in diminishedprotein-protein interaction.

Highly restrained cyclic peptide libraries are made by using codonswhich code mainly for amino acids with large side chains. That is, whenseveral resides of a cyclic peptide encode amino acids with large sidechains, the conformation space of the peptide is restricted. The resultis to bias the peptide to a higher affinity by reducing peptideconformational entropy. For example, a library of cyclic peptides couldbe created by restricting the triplet nucleotides coding for each randomamino acid in the library to C or T for the first position of thetriplet, A, G or T for the second position in the triplet, and G, C or Tfor the third position in the triplet. This would result in a librarybiased to large amino acids, i.e., phenylalanine (F), leucine (L),tyrosine (Y), histidine (H), glutamine (Q), cysteine (C), tryptophan (W)and arginine (R). A library biased toward large amino acid side chains,combined with the loss of glycine, alanine, serine, threonine,aspartate, and glutamate results in a library coding for more rigidpeptides. As this library lacks an acidic amino acid, a pre-synthesizedtriplet coding glutamate (i.e., GAG) or aspartate (GAC) may be addedduring the DNA synthesis of the library.

Alternatively, a large amino acid side chain residue library may becreated by pre-synthesizing triplets for desired residues. Theseresidues are then mixed together during the DNA synthesis of thelibrary. An example of a pre-synthesized large residue library is alibrary coding tyrosine (Y), arginine (R), glutamic acid (E), histidine(H), leucine (L), glutamine (Q), and optionally proline (P) or threonine(T).

A biased library can be created by restricting the number of conformersin a cyclic peptide. This approach is useful for structure activityrelationship optimization. The number of conformers may be restricted byfixing a proline in the cyclic peptide ring at one position and leavingall of the other residues random. A smaller number of conformers allowsfor higher affinity binding interactions with target molecules, and moreselective interactions with target moleucles due to a diminution of thepossibility of “induced fit” binding. “Induced fit” comes at the expenseof binding affinity due to a loss upon binding of the higherconformational entropy of a multi-conformer peptide. Higher affinity andselectivity are desirable for the development of cyclic peptides drugs.This is achieved by reducing the conformational entropy by including arigid amino acid in a fixed position in each library member. Forexample, fixing one proline in a 7 mer peptide is sufficient to restrictthe conformational space of the cyclic peptide. For 8 to 10 mers, twoprolines may be fixed in the ring allowing a diversity of (20)⁶ or6.4×10⁷ in the 6 unfixed position of a 10 mer ring. Such a library islarge enough to give hits in most screens for candidate drugs (asdescribed below).

As will be appreciated by those in the art, the type of cells used inthe present invention can vary widely. Basically, any mammalian cellsmay be used, with mouse, rat, primate and human cells being particularlypreferred, although as will be appreciated by those in the art,modifications of the system by pseudotyping allows all eukaryotic cellsto be used, preferably higher eukaryotes. As is more fully describedbelow, a screen will be set up such that the cells exhibit a selectablephenotype in the presence of a cyclic peptide. As is more fullydescribed below, cell types implicated in a wide variety of diseaseconditions are particularly useful, so long as a suitable screen may bedesigned to allow the selection of cells that exhibit an alteredphenotype as a consequence of the presence of a cyclic peptide withinthe cell.

Accordingly, suitable cell types include, but are not limited to, tumorcells of all types (particularly melanoma, myeloid leukemia, carcinomasof the lung, breast, ovaries, colon, kidney, prostate, pancreas andtestes), cardiomyocytes, endothelial cells, epithelial cells,lymphocytes (T-cell and B cell), mast cells, eosinophils, vascularintimal cells, hepatocytes, leukocytes including mononuclear leukocytes,stem cells such as haemopoetic, neural, skin, lung, kidney, liver andmyocyte stem cells (for use in screening for differentiation andde-differentiation factors), osteoclasts, chondrocytes and otherconnective tissue cells, keratinocytes, melanocytes, liver cells, kidneycells, and adipocytes. Suitable cells also include known research cells,including, but not limited to, Jurkat T cells, NIH3T3 cells, CHO, Cos,etc. See the ATCC cell line catalog, hereby expressly incorporated byreference.

The skilled artisan will appreciate that, in certain applications of thepresent invention, non-mammalian cells may be used. In particular, yeastcells may be employed

In one embodiment, the cells may be genetically engineered, that is,contain exogenous nucleic acid, for example, to contain targetmolecules.

Once made, the compositions of the invention find use in a number ofapplications. In particular, compositions with altered cyclizationefficiency are made. The compositions of the invention also may be usedto: (1) alter cellular phenotypes and/or physiology; (2) used inscreening assays to identify target molecules associated with changes incellular phenotype or phyisology; (3) used to inhibit protein-proteininteractions; (4) used as drugs to treat a number of disease states,such as cancer, cardiovascular diseases, obesity, neurologicaldisorders, etc.; and (5) used as drug leads to develop drugs to treatdisease states.

In a preferred embodiment, inteins with altered cyclization activity aregenerated. Naturally occurring inteins are mutagenized and tested invivo to determine whether the modified intein can catalyze protein orpeptide cyclization in mammalian cells: Preferably, inteins so modifiedare characterized by more efficient cyclization kinetics in vivo or bythe expression level of intein catalyzed cyclization scaffolds.Additional rounds of mutagenesis may be done to optimize in vivofunction. Assays useful for measuring intein-catalyzed cyclizationefficiency include fluorescent or gel-based assays directly measuringcyclic peptide or protein levels, and functional assays based on theproduction of a functional cyclic peptide whose effects can be measuredor selected for.

In a preferred embodiment, random mutagenesis (e.g., M13 primermutagenesis and PCR mutagenesis), PCR shuffling or other directedevolution techniques are directed to a target codon or region and theresulting intein variants screened for altered cyclization activity.These techniques are well known and can be directed to predeterminedsites, e.g., intein open reading frame or more specific regions orcodons within.

Amino acid substitutions are typically of single residues; insertionsusually will be on the order of from about 1 to 20 amino acids, althoughconsiderably larger insertions may be tolerated. Deletions range fromabout 1 to about 20 residues, although in some cases deletions may bemuch larger.

Substitutions, deletions, insertions or any combination thereof may beused to arrive at a final derivative. Generally these changes are doneon a few amino acids to minimize the alteration of the molecule.However, larger changes may be tolerated in certain circumstances. Whensmall alterations in the characteristics of the intein protein aredesired, substitutions are generally made in accordance with thefollowing chart: CHART 1 Original Exemplary Residue Substitutions AlaSer Arg Lys Asn Gln, His Asp Glu Cys Ser Gln Asn Glu Asp Gly Pro HisAsn, Gln Ile Leu, Val Leu Ile, Val Lys Arg, Gln, Glu Met Leu, Ile PheMet, Leu, Tyr Ser Thr Thr Ser Trp Tyr Tyr Trp, Phe Val Ile, Leu

Substantial changes in function are made by selecting substitutions thatare less conservative than those shown in Chart I. For example,substitutions may be made which more significantly affect: the structureof the polypeptide backbone in the area of the alteration, for examplethe alpha-helical or beta-sheet structure; the charge or hydrophobicityof the molecule at the target site; or the bulk of the side chain. Thesubstitutions which in general are expected to produce the greatestchanges in the polypeptide's properties are those in which (a) ahydrophilic residue, e.g., seryl or threonyl, is substituted for (or by)a hydrophobic residue, e.g., leucyl, isoleucyl, phenylalanyl, valyl oralanyl; (b) a cysteine or proline is substituted for (or by) any otherresidue; (c) a residue having an electropositive side chain, e.g.,lysyl, arginyl, or histidyl, is substituted for (or by) anelectronegative residue, e.g., glutamyl or aspartyl; or (d) a residuehaving a bulky side chain, e.g., phenylalanine, is substituted for (orby) one not having a side chain, e.g., glycine.

As outlined above, the variants typically exhibit the same qualitativebiological activity (i.e., cyclization) although variants may beselected to modify other characteristics of the intein protein asneeded. For example, endoplasmic reticulum/golgi directed inteinlibraries may be designed to operate in cellular environments moreacidic than the cytoplasmic compartment.

In a preferred embodiment specific residues of an intein motif aresubstituted, resulting in proteins with modified characteristics. Suchsubstitutions may occur at one or more residues, with 1-10 substitutionsbeing preferred. Preferred characteristics to be modified includecyclization efficiency, half-life, stability, and temperaturesensitivity.

In a preferred embodiment, intein mutants are generated using PCRmutagenesis. The resulting mutants are screened for altered cyclizationactivity. By “altered” cyclization activity” refers to anycharacteristic or attribute of an intein that can be selected ordetected and compared to the corresponding property of a naturallyoccurring intein. These properties include cyclization efficiency,stability, etc. Cyclization efficiency may be affected by the presenceor absence of a given amino acid, the size of the peptide library, etc.Unless otherwise specified, “altered” cyclization activity, whencomparing the cyclization efficiency of a mutant intein to thecyclization efficiency of wild-type or naturally occurring intein ispreferably at least 1-fold, more preferably at least a 10-fold increasein activity.

Screens for mutants with improved cyclization efficiency can be done inprocaryotes or eucaryotes. The mutants may be screened directly byassaying for the production of a cyclic peptide or indirectly byassaying a cyclic peptide's effects on a cell. Alternatively, themutants may be screened indirectly by assaying the product of the cyclicpeptide protein in vitro, e.g., enzyme inhibition assays, etc.

If the mutation prevents self-excision, no fluorescence is detected dueto the interruption in the tertiary structure of GFP. If the mutationdoes not effect self-excision or enhances splicing efficiency, thedegree of fluorescence may be quantified using a FACS analysis or othertechniques known in the art. In addition, cyclization of the GFPreconstitutes the myc epitope which can be detected using Westernanalysis.

In a preferred embodiment, a first plurality of cells is screened. Thatis, the cells into which the fusion nucleic acids are introduced arescreened for an altered phenotype. Thus, in this embodiment, the effectof the bioactive peptide is seen in the same cells in which it is made;i.e., an autocrine effect.

By a “plurality of cells” herein is meant roughly from about 10³ cellsto 10⁸ or 10⁹, with from 10⁶ to 10⁸ being preferred. This plurality ofcells comprises a cellular library, wherein generally each cell withinthe library contains a member of the peptide molecular library, i.e., adifferent peptide (or nucleic acid encoding the peptide), although aswill be appreciated by those in the art, some cells within the librarymay not contain a peptide, and some may contain more than species ofpeptide. When methods other than retroviral infection are used tointroduce the candidate nucleic acids into a plurality of cells, thedistribution of candidate nucleic acids within the individual cellmembers of the cellular library may vary widely, as it is generallydifficult to control the number of nucleic acids which enter a cellduring electroporation, etc.

In a preferred embodiment, the fusion nucleic acids are introduced intoa first plurality of cells, and the effect of the peptide is screened ina second or third plurality of cells, different from the first pluralityof cells, i.e., generally a different cell type. That is, the effect ofthe bioactive peptide is due to an extracellular effect on a secondcell, i.e., an endocrine or paracrine effect. This is done usingstandard techniques. The first plurality of cells may be grown in or onone media, and the media is allowed to touch a second plurality ofcells, and the effect measured. Alternatively, there may be directcontact between the cells. Thus, “contacting” is functional contact, andincludes both direct and indirect. In this embodiment, the firstplurality of cells may or may not be screened.

If necessary, the cells are treated to conditions suitable for theexpression of the peptide (for example, when inducible promoters areused).

Thus, the methods of the present invention comprise introducing amolecular library of fusion nucleic acids encoding randomized peptidesfused to scaffold into a plurality of cells, a cellular library. Each ofthe nucleic acids comprises a different nucleotide sequence encodingscaffold with a random peptide. The plurality of cells is then screened,as is more fully outlined below, for a cell exhibiting an alteredphenotype. The altered phenotype is due to the presence of a bioactivepeptide.

By “altered phenotype” or “changed physiology” or other grammaticalequivalents herein is meant that the phenotype of the cell is altered insome way, preferably in some detectable and/or measurable way. As willbe appreciated in the art, a strength of the present invention is thewide variety of cell types and potential phenotypic changes which may betested using the present methods. Accordingly, any phenotypic changewhich may be observed, detected, or measured may be the basis of thescreening methods herein. Suitable phenotypic changes include, but arenot limited to: gross physical changes such as changes in cellmorphology, cell growth, cell viability, adhesion to substrates or othercells, and cellular density; changes in the expression of one or moreRNAs, proteins, lipids, hormones, cytokines, or other molecules; changesin the equilibrium state (i.e., half-life) or one or more RNAs,proteins, lipids, hormones, cytokines, or other molecules; changes inthe localization of one or more RNAs, proteins, lipids, hormones,cytokines, or other molecules; changes in the bioactivity or specificactivity of one or more RNAs, proteins, lipids, hormones, cytokines,receptors, or other molecules; changes in the secretion of ions,cytokines, hormones, growth factors, or other molecules; alterations incellular membrane potentials, polarization, integrity or transport;changes in infectivity, susceptability, latency, adhesion, and uptake ofviruses and bacterial pathogens; etc. By “capable of altering thephenotype” herein is meant that the bioactive peptide can change thephenotype of the cell in some detectable and/or measurable way.

The altered phenotype may be detected in a wide variety of ways, as isdescribed more fully below, and will generally depend and correspond tothe phenotype that is being changed. Generally, the changed phenotype isdetected using, for example: microscopic analysis of cell morphology;standard cell viability assays, including both increased cell death andincreased cell viability, for example, cells that are now resistant tocell death via virus, bacteria, or bacterial or synthetic toxins;standard labeling assays such as fluorometric indicator assays for thepresence or level of a particular cell or molecule, including FACS orother dye staining techniques; biochemical detection of the expressionof target compounds after killing the cells; etc. In some cases, as ismore fully described herein, the altered phenotype is detected in thecell in which the fusion nucleic acid was introduced; in otherembodiments, the altered phenotype is detected in a second cell which isresponding to some molecular signal from the first cell.

An altered phenotype of a cell indicates the presence of a bioactivepeptide, acting preferably in a transdominant way. By “transdominant”herein is meant that the bioactive peptide indirectly causes the alteredphenotype by acting on a second molecule, which leads to an alteredphenotype. That is, a transdominant expression product has an effectthat is not in cis, i.e., a trans event as defined in genetic terms orbiochemical terms. A transdominant effect is a distinguishable effect bya molecular entity (i.e., the encoded peptide or RNA) upon some separateand distinguishable target; that is, not an effect upon the encodedentity itself. As such, transdominant effects include many well-knowneffects by pharmacologic agents upon target molecules or pathways incells or physiologic systems; for instance, the β-lactam antibioticshave a transdominant effect upon peptidoglycan synthesis in bacterialcells by binding to penicillin binding proteins and disrupting theirfunctions. An exemplary transdominant effect by a peptide is the abilityto inhibit NF—KB signaling by binding to IKB-α at a region critical forits function, such that in the presence of sufficient amounts of thepeptide (or molecular entity), the signaling pathways that normally leadto the activation of NF—KB through phosphorylation and/or degradation ofIKB-α are inhibited from acting at IKB-α because of the binding of thepeptide or molecular entity. In another instance, signaling pathwaysthat are normally activated to secrete IgE are inhibited in the presenceof peptide. Alternatively, signaling pathways in adipose tissue cells,normally quiescent, are activated to metabolize fat. In yet a furtheraspect, intracellular mechanisms for the replication of certain viruses,such as HIV-I, or Herpes viridae family members, or Respiratory SyncytiaVirus, for example, are inhibited in the presence of a peptide.

A transdominant effect upon a protein or molecular pathway is clearlydistinguishable from randomization, change, or mutation of a sequencewithin a protein or molecule of known or unknown function to enhance ordiminish a biochemical ability that protein or molecule alreadymanifests. For instance, a protein that enzymatically cleaves β-lactamantibiotics, a β-lactamase, could be enhanced or diminished in itsactivity by mutating sequences internal to its structure that enhance ordiminish the ability of this enzyme to act upon and cleave β-lactamantibiotics. This would be called a cis mutation to the protein. Theeffect of this protein upon β-lactam antibiotics is an activity theprotein already manifests, to a distinguishable degree. Similarly, amutation in the leader sequence that enhanced the export of this proteinto the extracellular spaces wherein it might encounter β-lactammolecules more readily, or a mutation within the sequence that enhancethe stability of the protein, would be termed cis mutations in theprotein. For comparison, a transdominant effector of this protein wouldinclude an agent, independent of the β-lactamase, that bound to theβ-lactamase in such a way that it enhanced or diminished the function ofthe β-lactamase by virtue of its binding to β-lactamase.

In a preferred embodiment, once a cell with an altered phenotype isdetected, the presence of the fusion protein is verified to ensure thatthe peptide was expressed and that the altered phenotype is due topresence of the peptide. As will be appreciated by those in the art,this verification of the presence of the peptide can be done eitherbefore, during or after the screening for an altered phenotype. This canbe done in a variety of ways, although preferred methods utilize FACStechniques.

In a preferred embodiment, the devices of the invention comprise liquidhandling components, including components for loading and unloadingfluids at each station or sets of stations. The liquid handling systemscan include robotic systems comprising any number of components. Inaddition, any or all of the steps outlined herein may be automated;thus, for example, the systems may be completely or partially automated.

As will be appreciated by those in the art, there are a wide variety ofcomponents which can be used, including, but not limited to, one or morerobotic arms; plate handlers for the positioning of microplates; holderswith cartridges and/or caps; automated lid or cap handlers to remove andreplace lids for wells on non-cross contamination plates; tip assembliesfor sample distribution with disposable tips; washable tip assembliesfor sample distribution; 96 well loading blocks; cooled reagent racks;microtitler plate pipette positions (optionally cooled); stacking towersfor plates and tips; and computer systems.

Fully robotic or microfluidic systems include automated liquid-,particle-, cell- and organism-handling including high throughputpipetting to perform all steps of screening applications. This includesliquid, particle, cell, and organism manipulations such as aspiration,dispensing, mixing, diluting, washing, accurate volumetric transfers;retrieving, and discarding of pipet tips; and repetitive pipetting ofidentical volumes for multiple deliveries from a single sampleaspiration. These manipulations are cross-contamination-free liquid,particle, cell, and organism transfers. This instrument performsautomated replication of microplate samples to filters, membranes,and/or daughter plates, high-density transfers, full-plate serialdilutions, and high capacity operation.

In a preferred embodiment, chemically derivatized particles, plates,cartridges, tubes, magnetic particles, or other solid phase matrix withspecificity to the assay components are used. The binding surfaces ofmicroplates, tubes or any solid phase matrices include non-polarsurfaces, highly polar surfaces, modified dextran coating to promotecovalent binding, antibody coating, affinity media to bind fusionproteins or peptides, surface-fixed proteins such as recombinant proteinA or G, nucleotide resins or coatings, and other affinity matrix areuseful in this invention.

In a preferred embodiment, platforms for multi-well plates, multi-tubes,holders, cartridges, minitubes, deep-well plates, microfuge tubes,cryovials, square well plates, filters, chips, optic fibers, beads, andother solid-phase matrices or platform with various volumes areaccommodated on an upgradable modular platform for additional capacity.This modular platform includes a variable speed orbital shaker, andmulti-position work decks for source samples, sample and reagentdilution, assay plates, sample and reagent reservoirs, pipette tips, andan active wash station.

In a preferred embodiment, thermocycler and thermoregulating systems areused for stabilizing the temperature of the heat exchangers such ascontrolled blocks or platforms to provide accurate temperature controlof incubating samples from 4° C. to 100° C.; this is in addition to orin place of the station thermocontrollers.

In a preferred embodiment, interchangeable pipet heads (single ormulti-channel) with single or multiple magnetic probes, affinity probes,or pipetters robotically manipulate the liquid, particles, cells, andorganisms. Multi-well or multi-tube magnetic separators or platformsmanipulate liquid, particles, cells, and organisms in single or multiplesample formats.

In some embodiments, for example when electronic detection is not done,the instrumentation will include a detector, which can be a wide varietyof different detectors, depending on the labels and assay. In apreferred embodiment, useful detectors include a microscope(s) withmultiple channels of fluorescence; plate readers to provide fluorescent,ultraviolet and visible spectrophotometric detection with single anddual wavelength endpoint and kinetics capability, fluroescence resonanceenergy transfer (FRET), luminescence, quenching, two-photon excitation,and intensity redistribution; CCD cameras to capture and transform dataand images into quantifiable formats; and a computer workstation.

These instruments can fit in a sterile laminar flow or fume hood, or areenclosed, self-contained systems, for cell culture growth andtransformation in multi-well plates or tubes and for hazardousoperations. The living cells will be grown under controlled growthconditions, with controls for temperature, humidity, and gas for timeseries of the live cell assays. Automated transformation of cells andautomated colony pickers will facilitate rapid screening of desiredcells.

Flow cytometry or capillary electrophoresis formats can be used forindividual capture of magnetic and other beads, particles, cells, andorganisms.

The flexible hardware and software allow instrument adaptability formultiple applications. The software program modules allow creation,modification, and running of methods. The system diagnostic modulesallow instrument alignment, correct connections, and motor operations.The customized tools, labware, and liquid, particle, cell and organismtransfer patterns allow different applications to be performed. Thedatabase allows method and parameter storage. Robotic and computerinterfaces allow communication between instruments.

In a preferred embodiment, the robotic apparatus includes a centralprocessing unit which communicates with a memory and a set ofinput/output devices (e.g., keyboard, mouse, monitor, printer, etc.)through a bus. Again, as outlined below, this may be in addition to orin place of the CPU for the multiplexing devices of the invention. Thegeneral interaction between a central processing unit, a memory,input/output devices, and a bus is known in the art. Thus, a variety ofdifferent procedures, depending on the experiments to be run, are storedin the CPU memory.

These robotic fluid handling systems can utilize any number of differentreagents, including buffers, reagents, samples, washes, assay componentssuch as label probes, etc.

Once the presence of the fusion protein is verified, the cell with thealtered phenotype is generally isolated from the plurality which do nothave altered phenotypes. This may be done in any number of ways, as isknown in the art, and will in some instances depend on the assay orscreen. Suitable isolation techniques include, but are not limited to,FACS, lysis selection using complement, cell cloning, scanning byFluorimager, expression of a “survival” protein, induced expression of acell surface protein or other molecule that can be rendered fluorescentor taggable for physical isolation; expression of an enzyme that changesa non-fluorescent molecule to a fluorescent one; overgrowth against abackground of no or slow growth; death of cells and isolation of DNA orother cell vitality indicator dyes, etc.

In a preferred embodiment, the fusion nucleic acid and/or the bioactivepeptide (i.e., the fusion protein) is isolated from the positive cell.This may be done in a number of ways. In a preferred embodiment, primerscomplementary to DNA regions common to the retroviral constructs, or tospecific components of the library such as a rescue sequence, definedabove, are used to “rescue” the unique random sequence. Alternatively,the fusion protein is isolated using a rescue sequence. Thus, forexample, rescue sequences comprising epitope tags or purificationsequences may be used to pull out the fusion protein usingimmunoprecipitation or affinity columns. In some instances, as isoutlined below, this may also pull out the primary target molecule ifthere is a sufficiently strong binding interaction between the bioactivepeptide and the target molecule. Alternatively, the peptide may bedetected using mass spectroscopy.

Once rescued, the sequence of the bioactive peptide and/or fusionnucleic acid is determined. This information can then be used in anumber of ways.

In a preferred embodiment, the bioactive peptide is resynthesized andreintroduced into the target cells to verify the effect. This may bedone using retroviruses, or alternatively using fusions to the HIV-1 Tatprotein, and analogs and related proteins, which allows very high uptakeinto target cells. See for example, Fawell et al., Proc. Natl. Acad.Sci. USA 91:664 (1994); Frankel et al., Cell 55:1189 (1988); Savion etal., J. Biol. Chem. 256:1149 (1981); Derossi et al., J. Biol. Chem.269:10444 (1994); and Baldin et al., EMBO J. 9:1511 (1990), all of whichare incorporated by reference.

In a preferred embodiment, the sequence of a bioactive peptide is usedto generate more candidate peptides. For example, the sequence of thebioactive peptide may be the basis of a second round of (biased)randomization, to develop bioactive peptides with increased or alteredactivities. Alternatively, the second round of randomization may changethe affinity of the bioactive peptide. Furthermore, it may be desirableto put the identified random region of the bioactive peptide into otherpresentation structures, or to alter the sequence of the constant regionof the presentation structure, to alter the conformation/shape of thebioactive peptide. It may also be desirable to “walk” around a potentialbinding site, in a manner similar to the mutagenesis of a bindingpocket, by keeping one end of the ligand region constant and randomizingthe other end to shift the binding of the peptide around.

In a preferred embodiment, either the bioactive peptide or the bioactivenucleic acid encoding it is used to identify target molecules, i.e., themolecules with which the bioactive peptide interacts. As will beappreciated by those in the art, there may be primary target moleculesto which the bioactive peptide binds or acts upon directly, and theremay be secondary target molecules, which are part of the signallingpathway affected by the bioactive peptide; these might be termed“validated targets”.

In a preferred embodiment, the bioactive peptide is a drug. As will beappreciated by those in the art, the structure of the cyclic peptide maybe modeled and used in rational drug design to synthesize agents thatmimic the interaction of the cyclic peptide with its' target. Drugs mayalso be modeled based on the three dimensional structure of the peptidebound to its target. Drugs so modeled may have structures that aresimilar to or unrelated to the starting structure of the cyclic peptideor the cyclic peptide bound to its target. Alternatively, highthroughput screens can be used to identify small molecules capable ofcompeting with the cyclic peptide for its target.

In a preferred embodiment, the bioactive cyclic peptide may be used asthe starting point for designing/synthesizing derivative molecules withsimilar or more favorable properties for use as a drug. For example,individual amino acids, specific chemical groups, etc., can be replacedand the derivative molecule tested for use as a drug. Both naturallyoccurring and synthetic amino acid analogs (see below for definition)can be introduced in to the derivative molecule to optimize propertiessuch as binding, stability, and pharmocokinectics. Preferably, thederivative molecule has one or more of the following properties:improved stability, higher binding affinity, improved specificity forthe target, improved pharmocokinetics, e.g., absorption, distribution,resistance to degradation, etc.

In a preferred embodiment, the bioactive peptide is used to pull outtarget molecules. For example, as outlined herein, if the targetmolecules are proteins, the use of epitope tags, purification sequences,or affinity tags can allow the purification of primary target moleculesvia biochemical means (co-immunoprecipitation, affinity columns, etc.).Alternatively, the peptide, when expressed in bacteria and purified, canbe used as a probe against a bacterial cDNA expression library made frommRNA of the target cell type. Or, peptides can be used as “bait” ineither yeast or mammalian two or three hybrid systems. Such interactioncloning approaches have been very useful to isolate DNA-binding proteinsand other interacting protein components. The peptide(s) can be combinedwith other pharmacologic activators to study the epistatic relationshipsof signal transduction pathways in question. It is also possible tosynthetically prepare labeled peptide and use it to screen a cDNAlibrary expressed in bacteriophage for those cDNAs which bind thepeptide. Furthermore, it is also possible that one could use cDNAcloning via retroviral libraries to “complement” the effect induced bythe peptide. In such a strategy, the peptide would be required to bestochiometrically titrating away some important factor for a specificsignaling pathway. If this molecule or activity is replenished byover-expression of a cDNA from within a cDNA library, then one can clonethe target. Similarly, cDNAs cloned by any of the above yeast orbacteriophage systems can be reintroduced to mammalian cells in thismanner to confirm that they act to complement function in the system thepeptide acts upon.

In a preferred embodiment, target molecules are identified byincorporating an affinity tagged amino acid residue into the sequence ofthe cyclic peptide. For example, incorporation of a cysteine alows forthe chemical conjugation of the cyclic peptide to a solid support matrixvia a disulfide bond. In particular, target molecules that bind tofunctional cyclic peptides are isolated and identified using affinitytagged amino acids.

In a preferred embodiment, the cysteine contributed by the extein isuniquely alkylated with an affinity reagent as part of the synthesis ofthe peptide to allow affinity extraction and identification of targetmolecules using HPLC-mass spectrometry methods. Cysteine-alkylatedcyclic peptide analogs are tested for function, and if functional,target molecules are affinity extracted using methods well known in theart. If the cysteine-alkylated peptide analogs are not functional,synthetic cyclic peptide analogs are constructed with cysteine-affinitytag amino acid analogs in other positions and tested for function. Inalternative embodiments, lysine affinity tagged amino acids are used.

Alternatively, if an affinity tagged amino acid cannot be produced invivo, the tag can be introduced in vitro and tested in vivo forfunction.

Any amino acid which can be used as an affinity tag may be used in themethods of the invention. This includes both naturally occurring andsynthetic amino acid analogs which can be introduced into the cyclicpeptide to facilitate chemical conjugation or binding to a solid supportmatrix. Thus “amino acid”, or “peptide residue”, as used herein meansboth naturally occurring and synthetic amino acids. For example,homo-phenylalanine, citrulline, and noreleucine are considered aminoacids for the purposes of the invention. “Amino acid” also includesimino acid residues such as proline and hydroxyproline. In addition, anyamino acid can be replaced by the same amino acid but of the oppositechirality. Thus, any amino acid naturally occurring in theL-configuration (which may also be referred to as the R or S, dependingupon the structure of the chemical entity) may be replaced with an aminoacid of the same chemical structural type, but of the oppositechirality, generally referred to as the D-amino acid but which canadditionally be referred to as the R— or the S—, depending upon itscomposition and chemical configuration. Such derivatives have theproperty of greatly increased stability, and therefore are advantageousin the formulation of compounds which may have longer in vivo halflives, when administered by oral, intravenous, intramuscular,intraperitoneal, topical, rectal, intraocular, or other routes.

In the preferred embodiment, the amino acids are in the (S) orL-configuration. If non-naturally occurring side chains are used,non-amino acid substituents may be used, for example to prevent orretard in vivo degradations. Proteins including non-naturally occurringamino acids may be synthesized or in some cases, made recombinantly; seevan Hest et al., FEBS Lett 428:(1-2) 68-70 (1998) and Tang et al.,Abstr. Pap Am. Chem. S218:U138-U138 Part 2 Aug. 22, 1999, both of whichare expressly incorporated by reference herein.

Aromatic amino acids may be replaced with D- or L-naphylalanine, D- orL-Phenylglycine, D- or L-2-thieneylalanine, D- or L-1-, 2-, 3- or4-pyreneylalanine, D- or L-3-thieneylalanine, D- orL-(2-pyridinyl)-alanine, D- or L-(3-pyridinyl)-alanine, D- orL-(2-pyrazinyl)-alanine, D- or L-(4-isopropyl)-phenylglycine,D-(trifluoromethyl)-phenylglycine, D-(trifluoromethyl)-phenylalanine,D-p-fluorophenylalanine, D- or L-p-biphenylphenylalanine, D- orL-p-methoxybiphenylphenylalanine, D- or L-2-indole(alkyl)alanines, andD- or L-alkylainines where alkyl may be substituted or unsubstitutedmethyl, ethyl, propyl, hexyl, butyl, pentyl, isopropyl, iso-butyl,sec-isotyl, iso-pentyl, non-acidic amino acids, of C1-C20. Acidic aminoacids can be substituted with non-carboxylate amino acids whilemaintaining a negative charge, and derivatives or analogs thereof, suchas the non-limiting examples of (phosphono)alanine, glycine, leucine,isoleucine, threonine, or serine; or sulfated (e.g., —SO.sub.3 H)threonine, serine, tyrosine. Other substitutions may include unnaturalhyroxylated amino acids may made by combining “alkyl” with any naturalamino acid. The term “alkyl” as used herein refers to a branched orunbranched saturated hydrocarbon group of 1 to 24 carbon atoms, such asmethyl, ethyl, n-propyl, isoptopyl, n-butyl, isobutyl, t-butyl, octyl,decyl, tetradecyl, hexadecyl, eicosyl, tetracisyl and the like. Alkylincludes heteroalkyl, with atoms of nitrogen, oxygen and sulfur.Preferred alkyl groups herein contain 1 to 12 carbon atoms. Basic aminoacids may be substituted with alkyl groups at any position of thenaturally occurring amino acids lysine, arginine, ornithine, citrulline,or (guanidino)-acetic acid, or other (guanidino)alkyl-acetic acids,where “alkyl” is define as above. Nitrile derivatives (e.g., containingthe CN-moiety in place of COOH) may also be substituted for asparagineor glutamine, and methionine sulfoxide may be substituted formethionine. Methods of preparation of such peptide derivatives are wellknown to one skilled in the art.

In addition, any amide linkage can be replaced by a ketomethylenemoiety. Such derivatives are expected to have the property of increasedstability to degradation by enzymes, and therefore possess advantagesfor the formulation of compounds which may have increased in vivo halflives, as administered by oral, intravenous, intramuscular,intraperitoneal, topical, rectal, intraocular, or other routes.Additional amino acid modifications of amino acids of to the presentinvention may include the following: Cysteinyl residues may be reactedwith alpha-haloacetates (and corresponding amines), such as2-chloroacetic acid or chloroacetamide, to give carboxymethyl orcarboxyamidomethyl derivatives. Cysteinyl residues may also bederivatized by reaction with compounds such as bromotrifluoroacetone,alpha-bromo-beta-(5-imidozoyl)propionic acid, chloroacetyl phosphate,N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyldisulfide, p-chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, orchloro-7-nitrobenzo-2-oxa-1,3-diazole. Histidyl residues may bederivatized by reaction with compounds such as diethylprocarbonate e.g.,at pH 5.5-7.0 because this agent is relatively specific for the histidylside chain, and para-bromophenacyl bromide may also be used; e.g., wherethe reaction is preferably performed in 0.1 M sodium cacodylate at pH6.0. Lysinyl and amino terminal residues may be reacted with compoundssuch as succinic or other carboxylic acid anhydrides. Derivatizationwith these agents is expected to have the effect of reversing the chargeof the lysinyl residues. Other suitable reagents for derivatizingalpha-amino-containing residues include compounds such as imidoesters,e.g., as methyl picolinimidate; pyridoxal phosphate; pyridoxal;chloroborohydride; trinitrobenzenesulfonic acid; O-methylisourea; 2,4pentanedione; and transaminase-catalyzed reaction with glyoxylate.Arginyl residues may be modified by reaction with one or severalconventional reagents, among them phenylglyoxal, 2,3-butanedione,1,2-cyclohexanedione, and ninhydrin according to known method steps.Derivatization of arginine residues requires that the reaction beperformed in alkaline conditions because of the high pKa of theguanidine functional group. Furthermore, these reagents may react withthe groups of lysine as well as the arginine epsilon-amino group. Thespecific modification of tyrosyl residues per se is well-known, such asfor introducing spectral labels into tyrosyl residues by reaction witharomatic diazonium compounds or tetranitromethane. N-acetylimidizol andtetranitromethane may be used to form O-acetyl tyrosyl species and3-nitro derivatives, respectively. Carboxyl side groups (aspartyl orglutamyl) may be selectively modified by reaction with carbodiimides(R′—N—C—N—R′) such as 1-cyclohexyl-3-(2-morpholinyl-(4-ethyl)carbodiimide or 1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide.Furthermore aspartyl and glutamyl residues may be converted toasparaginyl and glutaminyl residues by reaction with ammonium ions.Glutaminyl and asparaginyl residues may be frequently deamidated to thecorresponding glutamyl and aspartyl residues. Alternatively, theseresidues may be deamidated under mildly acidic conditions. Either formof these residues falls within the scope of the present invention.

Examples of affinity labeled amino acids useful for extraction of targetmolecules include lysine-epsilon amino biotin, or lysine reacted withamine-specific biotinylation reagents such as biotin-NHS ester andsulfo-NHS biotin.

Spacers may be incorporated between the affinity element and the peptideto relieve steric restraints between the affinity tag and a cyclicpeptide bound to a target molecule. A spacer which may be used withaffinity tagged lysine is NHS-LC-biotin (Pierce Checmical CO., RockfordIll.), although other spacers as are known in the art also may be used.

Examples of spacers which can be used with affinity tagged cysteinesinclude cysteine reacted with iodoacetamido-biotin,biotin-hexyl-3′-(2′-pyridyldithio) propionamide (a 29 Å spacer fromPierce Chemical), iodoacetyl-LC-biotin (27 Å spacer) or biotin-BMCC witha 32 Å spacer (Pierce Chemical). An example of a spacer used withaffinity tagged cysteine is shown in Structure 1:

Alternatively, as part of the solid phase synthesis of the peptide,affinity tags may be synthesized branching off from the cysteine orlysine. In this case, the spacer consists of a defined number (i.e., n)of amino acids branching off the side chain of the cysteine or lysine oranother residue of the cyclic peptide. Preferably, n=1 to 40. Thisallows for spacers of variable length, ranging from 3 Å to 100 Å ormore. Gycines, because of their flexibility, are preferred because asterically bulky target molecules bound to the cyclic peptide can beaccommodated. The affinity tag is inserted at the end of the side chainas illustrated in Structure 2:

In a preferred embodiment, the spacer is at least one protein diameterlong (20-40 Å). When the interacting target molecule is part of a largecomplex, the spacer is up to at least two protein diameters (40-80 Å).

Once primary target molecules have been identified, secondary targetmolecules may be identified in the same manner, using the primary targetas the “bait”. In this manner, signaling pathways may be elucidated.Similarly, bioactive peptides specific for secondary target moleculesmay also be discovered, to allow a number of bioactive peptides to acton a single pathway, for example for combination therapies.

The screening methods of the present invention may be useful to screen alarge number of cell types under a wide variety of conditions.Generally, the host cells are cells that are involved in disease states,and they are tested or screened under conditions that normally result inundesirable consequences on the cells. When a suitable bioactive peptideis found, the undesirable effect may be reduced or eliminated.Alternatively, normally desirable consequences may be reduced oreliminated, with an eye towards elucidating the cellular mechanismsassociated with the disease state or signalling pathway.

In a preferred embodiment, the present methods are useful in cancerapplications. The ability to rapidly and specifically kill tumor cellsis a cornerstone of cancer chemotherapy. In general, using the methodsof the present invention, random libraries can be introduced into anytumor cell (primary or cultured), and peptides identified which bythemselves induce apoptosis, cell death, loss of cell division ordecreased cell growth. This may be done de novo, or by biasedrandomization toward known peptide agents, such as angiostatin, whichinhibits blood vessel wall growth. Alternatively, the methods of thepresent invention can be combined with other cancer therapeutics (e.g.,drugs or radiation) to sensitize the cells and thus induce rapid andspecific apoptosis, cell death, loss of cell division or decreased cellgrowth after exposure to a secondary agent. Similarly, the presentmethods may be used in conjunction with known cancer therapeutics toscreen for agonists to make the therapeutic more effective or lesstoxic. This is particularly preferred when the chemotherapeutic is veryexpensive to produce such as taxol.

Known oncogenes such as v-Abl, v-Src, v-Ras, and others, induce atransformed phenotype leading to abnormal cell growth when transfectedinto certain cells. This is also a major problem with micro-metastases.Thus, in a preferred embodiment, non-transformed cells can betransfected with these oncogenes, and then random libraries introducedinto these cells, to select for bioactive peptides which reverse orcorrect the transformed state. One of the signal features of oncogenetransformation of cells is the loss of contact inhibition and theability to grow in soft-agar. When transforming viruses are constructedcontaining v-Abl, v-Src, or v-Ras in IRES-puro retroviral vectors,infected into target 3T3 cells, and subjected to puromycin selection,all of the 3T3 cells hyper-transform and detach from the plate. Thecells may be removed by washing with fresh medium. This can serve as thebasis of a screen, since cells which express a bioactive peptide willremain attached to the plate and form colonies.

Similarly, the growth and/or spread of certain tumor types are enhancedby stimulatory responses from growth factors and cytokines (e.g., PDGF,EGF, Heregulin, and others) which bind to receptors on the surfaces ofspecific tumors. In a preferred embodiment, the methods of the inventionare used to inhibit or stop tumor growth and/or spread, by findingbioactive peptides capable of blocking the ability of the growth factoror cytokine to stimulate the tumor cell. For example, random librariesmay be introduced into specific tumor cells with the addition of thegrowth factor or cytokine, followed by selection of bioactive peptideswhich block the binding, signaling, phenotypic and/or functionalresponses of these tumor cells to the growth factor or cytokine inquestion.

Similarly, the spread of cancer cells (invasion and metastasis) is asignificant problem limiting the success of cancer therapies. Theability to inhibit the invasion and/or migration of specific tumor cellswould be a significant advance in the therapy of cancer. Tumor cellsknown to have a high metastatic potential (for example, melanoma, lungcell carcinoma, breast and ovarian carcinoma) can have random librariesintroduced into them, and peptides selected which in a migration orinvasion assay, inhibit the migration and/or invasion of specific tumorcells. Particular applications for inhibition of the metastaticphenotype, which could allow a more specific inhibition of metastasis,include the metastasis suppressor gene NM23, which codes for adinucleoside diphosphate kinase. Thus intracellular peptide activatorsof this gene could block metastasis, and a screen for its upregulation(by fusing it to a reporter gene) would be of interest. Many oncogenesalso enhance metastasis. Peptides which inactivate or counteract mutatedRAS oncogenes, v-MOS, v-RAF, A-RAF, v-SRC, v-FES, and v-FMS would alsoact as anti-metastatics. Peptides that act intracellularly to block therelease of combinations of proteases required for invasion, such as thematrix metalloproteases and urokinase, could also be effectiveantimetastatics.

In a preferred embodiment, the random libraries of the present inventionare introduced into tumor cells known to have inactivated tumorsuppressor genes, and successful reversal by either reactivation orcompensation of the knockout would be screened by restoration of thenormal phenotype. One example is the reversal of p53-inactivatingmutations, which are present in 50% or more of all cancers. Since p53'sactions are complex and involve its action as a transcription factor,there are probably numerous potential ways a peptide or small moleculederived from a peptide could reverse the mutation. For instance, onebasis would be upregulation of the immediately downstreamcyclin-dependent kinase p21CIP1/WAF1. To be useful such reversal wouldhave to work for many of the different known p53 mutations. This iscurrently being approached by gene therapy; one or more small moleculeswhich do this might be preferable.

Another example involves screening of bioactive peptides that restorethe constitutive function of the brca-1 or brca-2 genes, and other tumorsuppressor genes important in breast cancer, for example the adenomatouspolyposis coli gene (APC) and the Drosophila discs-large gene (Dlg),which are components of cell-cell junctions. Mutations of brca-1 areimportant in hereditary ovarian and breast cancers and constitute anadditional application of the present invention.

In a preferred embodiment, the methods of the present invention are usedto create novel cell lines from cancers from patients. A retrovirallydelivered short peptide which inhibits the final common pathway ofprogrammed cell death should allow for short- and possibly long-termcell lines to be established. Conditions of in vitro culture andinfection of human leukemia cells will be established. There is a realneed for methods which allow the maintenance of certain tumor cells inculture long enough to allow for physiological and pharmacologicalstudies. Currently, some human cell lines have been established by theuse of transforming agents such as Epstein-Barr virus that considerablyalters the existing physiology of the cell. On occasion, cells will growon their own in culture but this is a random event. Programmed celldeath (apoptosis) occurs via complex signaling pathways within cellsthat ultimately activate a final common pathway producing characteristicchanges in the cell leading to a non-inflammatory destruction of thecell. It is well known that tumor cells have a high apoptotic index, orpropensity to enter apoptosis in vivo. When cells are placed in culture,the in vivo stimuli for malignant cell growth are removed and cellsreadily undergo apoptosis. The objective would be to develop thetechnology to establish cell lines from any number of primary tumorcells, for example primary human leukemia cells, in a reproduciblemanner without altering the native configuration of the signalingpathways in these cells. By introducing nucleic acids encoding peptideswhich inhibit apoptosis, increased cell survival in vitro, and hence theopportunity to study signalling transduction pathways in primary humantumor cells, is accomplished. In addition, these methods may be used forculturing primary cells, i.e., non-tumor cells.

In a preferred embodiment, the present methods are useful incardiovascular applications. In a preferred embodiment, cardiomyocytesmay be screened for the prevention of cell damage or death in thepresence of normally injurious conditions, including, but not limitedto, the presence of toxic drugs (particularly chemotherapeutic drugs),for example, to prevent heart failure following treatment withadriamycin; anoxia, for example in the setting of coronary arteryocclusion; and autoimmune cellular damage by attack from activatedlymphoid cells (for example as seen in post viral myocarditis andlupus). Candidate bioactive peptides are inserted into cardiomyocytes,the cells are subjected to the insult, and bioactive peptides areselected that prevent any or all of: apoptosis; membrane depolarization(e.g., decrease arrythmogenic potential of insult); cell swelling; orleakage of specific intracellular ions, second messengers and activatingmolecules (for example, arachidonic acid and/or lysophosphatidic acid).

In a preferred embodiment, the present methods are used to screen fordiminished arrhythmia potential in cardiomyocytes. The screens comprisethe introduction of the candidate nucleic acids encoding candidatebioactive peptides, followed by the application of arrythmogenicinsults, with screening for bioactive peptides that block specificdepolarization of cell membrane. This may be detected using patchclamps, or via fluorescence techniques). Similarly, channel activity(for example, potassium and chloride channels) in cardiomyocytes couldbe regulated using the present methods in order to enhance contractilityand prevent or diminish arrhythmias.

In a preferred embodiment, the present methods are used to screen forenhanced contractile properties of cardiomyocytes and diminish heartfailure potential. The introduction of the libraries of the inventionfollowed by measuring the rate of change of myosinpolymerization/depolymerization using fluorescent techniques can bedone. Bioactive peptides which increase the rate of change of thisphenomenon can result in a greater contractile response of the entiremyocardium, similar to the effect seen with digitalis.

In a preferred embodiment, the present methods are useful to identifyagents that will regulate the intracellular and sarcolemmal calciumcycling in cardiomyocytes in order to prevent arrhythmias. Bioactivepeptides are selected that regulate sodium-calcium exchange, sodiumproton pump function, and regulation of calcium-ATPase activity.

In a preferred embodiment, the present methods are useful to identifyagents that diminish embolic phenomena in arteries and arteriolesleading to strokes (and other occlusive events leading to kidney failureand limb ischemia) and angina precipitating a myocardial infarct areselected. For example, bioactive peptides may be identified that willdiminish the adhesion of platelets and leukocytes, and thus diminish theocclusion events. Adhesion in this setting can be inhibited by thelibraries of the invention being inserted into endothelial cells(quiescent cells, or activated by cytokines, e.g., IL-1, and growthfactors, e.g., PDGF/EGF) and then screening for peptides that either: 1)down regulate adhesion molecule expression on the surface of theendothelial cells (binding assay); 2) block adhesion molecule activationon the surface of these cells (signaling assay); or 3) release in anautocrine manner peptides that block receptor binding to the cognatereceptor on the adhering cell.

Embolic phenomena can also be addressed by activating proteolyticenzymes on the cell surfaces of endothelial cells, and thus releasingactive enzyme which can digest blood clots. Thus, delivery of thelibraries of the invention to endothelial cells is done, followed bystandard fluorogenic assays, which will allow monitoring of proteolyticactivity on the cell surface towards a known substrate. Bioactivepeptides can then be selected which activate specific enzymes towardsspecific substrates.

In a preferred embodiment, arterial inflammation in the setting ofvasculitis and post-infarction can be regulated by decreasing thechemotactic responses of leukocytes and mononuclear leukocytes. This canbe accomplished by blocking chemotactic receptors and their respondingpathways on these cells. Candidate bioactive libraries can be insertedinto these cells, and the chemotactic response to diverse chemokines(for example, to the IL-8 family of chemokines, RANTES) inhibited incell migration assays.

In a preferred embodiment, arterial restenosis following coronaryangioplasty can be controlled by regulating the proliferation ofvascular intimal cells and capillary and/or arterial endothelial cells.Candidate bioactive peptide libraries can be inserted into these celltypes and their proliferation in response to specific stimuli monitored.One application may be intracellular peptides which block the expressionor function of c-myc and other oncogenes in smooth muscle cells to stoptheir proliferation. A second application may involve the expression oflibraries in vascular smooth muscle cells to selectively induce theirapoptosis. Application of small molecules derived from these peptidesmay require targeted drug delivery; this is available with stents,hydrogel coatings, and infusion-based catheter systems. Peptides whichdownregulate endothelin-IA receptors or which block the release of thepotent vasoconstrictor and vascular smooth muscle cell mitogenendothelin-1 may also be candidates for therapeutics. Peptides can beisolated from these libraries which inhibit growth of these cells, orwhich prevent the adhesion of other cells in the circulation known torelease autocrine growth factors, such as platelets (PDGF) andmononuclear leukocytes.

The control of capillary and blood vessel growth is an important goal inorder to promote increased blood flow to ischemic areas (growth), or tocut-off the blood supply (angiogenesis inhibition) of tumors. Candidatebioactive peptide libraries can be inserted into capillary endothelialcells and their growth monitored. Stimuli such as low oxygen tension andvarying degrees of angiogenic factors can regulate the responses, andpeptides isolated that produce the appropriate phenotype. Screening forantagonism of vascular endothelial cell growth factor, important inangiogenesis, would also be useful.

In a preferred embodiment, the present methods are useful in screeningfor decreases in atherosclerosis producing mechanisms to find peptidesthat regulate LDL and HDL metabolism. Candidate libraries can beinserted into the appropriate cells (including hepatocytes, mononuclearleukocytes, endothelial cells) and peptides selected which lead to adecreased release of LDL or diminished synthesis of LDL, or converselyto an increased release of HDL or enhanced synthesis of HDL. Bioactivepeptides can also be isolated from candidate libraries which decreasethe production of oxidized LDL, which has been implicated inatherosclerosis and isolated from atherosclerotic lesions. This couldoccur by decreasing its expression, activating reducing systems orenzymes, or blocking the activity or production of enzymes implicated inproduction of oxidized LDL, such as 15-lipoxygenase in macrophages.

In a preferred embodiment, the present methods are used in screens toregulate obesity via the control of food intake mechanisms ordiminishing the responses of receptor signaling pathways that regulatemetabolism. Bioactive peptides that regulate or inhibit the responses ofneuropeptide Y (NPY), cholecystokinin and galanin receptors, areparticularly desirable. Candidate libraries can be inserted into cellsthat have these receptors cloned into them, and inhibitory peptidesselected that are secreted in an autocrine manner that block thesignaling responses to galanin and NPY. In a similar manner, peptidescan be found that regulate the leptin receptor.

In a preferred embodiment, the present methods are useful inneurobiology applications. Candidate libraries may be used for screeningfor anti-apoptotics for preservation of neuronal function and preventionof neuronal death. Initial screens would be done in cell culture. Oneapplication would include prevention of neuronal death, by apoptosis, incerebral ischemia resulting from stroke. Apoptosis is known to beblocked by neuronal apoptosis inhibitory protein (NAIP); screens for itsupregulation, or effecting any coupled step could yield peptides whichselectively block neuronal apoptosis. Other applications includeneurodegenerative diseases such as Alzheimer's disease and Huntington'sdisease.

In a preferred embodiment, the present methods are useful in bonebiology applications. Osteoclasts are known to play a key role in boneremodeling by breaking down “old” bone, so that osteoblasts can lay down“new” bone. In osteoporosis one has an imbalance of this process.Osteoclast overactivity can be regulated by inserting candidatelibraries into these cells, and then screening for bioactive peptidesthat produce: 1) a diminished processing of collagen by these cells; 2)decreased pit formation on bone chips; and 3) decreased release ofcalcium from bone fragments.

The present methods may also be used to screen for agonists of bonemorphogenic proteins, hormone mimetics to stimulate, regulate, orenhance new bone formation (e.g., in a manner similar to parathyroidhormone and calcitonin). These have use in osteoporosis, for poorlyhealing fractures, and to accelerate the rate of healing of newfractures. Furthermore, cell lines of connective tissue origin can betreated with candidate libraries and screened for their growth,proliferation, collagen stimulating activity, and/or prolineincorporating ability on the target osteoblasts. Alternatively,candidate libraries can be expressed directly in osteoblasts orchondrocytes and screened for increased production of collagen or bone.

In a preferred embodiment, the present methods are useful in skinbiology applications. Keratinocyte responses to a variety of stimuli mayresult in psoriasis, a proliferative change in these cells. Candidatelibraries can be inserted into cells removed from active psoriaticplaques, and bioactive peptides isolated which decrease the rate ofgrowth of these cells.

In a preferred embodiment, the present methods are useful in theregulation or inhibition of keloid formation (i.e., excessive scarring).Candidate libraries inserted into skin connective tissue cells isolatedfrom individuals with this condition, and bioactive peptides isolatedthat decrease proliferation, collagen formation, or prolineincorporation: Results from this work can be extended to treat theexcessive scarring that also occurs in burn patients. If a commonpeptide motif is found in the context of the keloid work, then it can beused widely in a topical manner to diminish scarring post burn.

Similarly, wound healing for diabetic ulcers and other chronic “failureto heal” conditions in the skin and extremities can be regulated byproviding additional growth signals to cells which populate the skin anddermal layers. Growth factor mimetics may in fact be very useful forthis condition. Candidate libraries can be inserted into skin connectivetissue cells, and bioactive peptides isolated which promote the growthof these cells under “harsh” conditions, such as low oxygen tension, lowpH, and the presence of inflammatory mediators.

Cosmeceutical applications of the present invention include the controlof melanin production in skin melanocytes. For example, a naturallyoccurring peptide, arbutin, is a tyrosine hydroxylase inhibitor, a keyenzyme in the synthesis of melanin. Candidate libraries can be insertedinto melanocytes and known stimuli that increase the synthesis ofmelanin applied to the cells. Bioactive peptides can be isolated thatinhibit the synthesis of melanin under these conditions.

In a preferred embodiment, the present methods are useful inendocrinology applications. The retroviral peptide library technologycan be applied broadly to any endocrine, growth factor, cytokine orchemokine network which involves a signaling peptide or protein thatacts in either an endocrine, paracrine or autocrine manner that binds ordimerizes a receptor and activates a signaling cascade that results in aknown phenotypic or functional outcome. The methods are applied so as toisolate a peptide which either mimics the desired hormone (e.g.,insulin, leptin, calcitonin, PDGF, EGF, EPO, GMCSF, IL1-17, mimetics) orinhibits its action by either blocking the release of the hormone,blocking its binding to a specific receptor or carrier protein (forexample, CRF binding protein), or inhibiting the intracellular responsesof the specific target cells to that hormone. Selection of peptideswhich increase the expression or release of hormones from the cellswhich normally produce them could have broad applications to conditionsof hormonal deficiency.

In a preferred embodiment, the present methods are useful in infectiousdisease applications. Viral latency (herpes viruses such as CMV, EBV,HBV, and other viruses such as HIV) and their reactivation are asignificant problem, particularly in immunosuppressed patients (patientswith AIDS and transplant patients). The ability to block thereactivation and spread of these viruses is an important goal. Celllines known to harbor or be susceptible to latent viral infection can beinfected with the specific virus, and then stimuli applied to thesecells which have been shown to lead to reactivation and viralreplication. This can be followed by measuring viral titers in themedium and scoring cells for phenotypic changes. Candidate libraries canthen be inserted into these cells under the above conditions, andpeptides isolated which block or diminish the growth and/or release ofthe virus. As with chemotherapeutics, these experiments can also be donewith drugs which are only partially effective towards this outcome, andbioactive peptides isolated which enhance the virucidal effect of thesedrugs. Bioactive peptides may also be tested for the ability to blocksome aspect of viral assembly, viral replication, entry or infectiouscycle.

One example of many is the ability to block HIV-1 infection. HIV-1requires CD4 and a co-receptor which can be one of several seventransmembrane G-protein coupled receptors. In the case of the infectionof macrophages, CCR-5 is the required co-receptor, and there is strongevidence that a block on CCR-5 will result in resistance to HIV-1infection. There are two lines of evidence for this statement. First, itis known that the natural ligands for CCR-5, the CC chemokines RANTES,MIP1a and MIP1b are responsible for CD8+ mediated resistance to HIV.Second, individuals homozygous for a mutant allele of CCR-5 arecompletely resistant to HIV infection. Thus, an inhibitor of theCCR-5/HIV interaction would be of enormous interest to both biologistsand clinicians. The extracellular anchored constructs offer superb toolsfor such a discovery. Into the transmembrane, epitope tagged,glycine-serine tethered constructs (ssTM V G20 E TM), one can place arandom, cyclized peptide library of the general sequence CNNNNNNNNNNC orC—(X)_(n)—C. Then one infects a cell line that expresses CCR-5 withretroviruses containing this library. Using an antibody to CCR-5 one canuse FACS to sort desired cells based on the binding of this antibody tothe receptor. All cells which do not bind the antibody will be assumedcontain inhibitors of this antibody binding site. These inhibitors, inthe retroviral construct can be further assayed for their ability toinhibit HIV-1 entry.

Viruses are known to enter cells using specific receptors to bind tocells (for example, HIV uses CD4, coronavirus uses CD13, murine leukemiavirus uses transport protein, and measles virus uses CD44) and to fusewith cells (HIV uses chemokine receptor). Candidate libraries can beinserted into target cells known to be permissive to these viruses, andbioactive peptides isolated which block the ability of these viruses tobind and fuse with specific target cells.

Intein libraries may also be used to screen for cyclic peptides whichblock HIV-1 infection. For example, inteins can be designed such thatcyclized peptides are secreted from cells where they can bind to CCR5and antagonize HIV-1 binding.

In a preferred embodiment, the present invention finds use withinfectious organisms. Intracellular organisms such as mycobacteria,listeria, salmonella, pneumocystis, yersinia, leishmania, T. cruzi, canpersist and replicate within cells, and become active inimmunosuppressed patients. There are currently drugs on the market andin development which are either only partially effective or ineffectiveagainst these organisms. Candidate libraries can be inserted intospecific cells infected with these organisms (pre- or post-infection),and bioactive peptides selected which promote the intracellulardestruction of these organisms in a manner analogous to intracellular“antibiotic peptides” similar to magainins. In addition peptides can beselected which enhance the cidal properties of drugs already underinvestigation which have insufficient potency by themselves, but whencombined with a specific peptide from a candidate library, aredramatically more potent through a synergistic mechanism. Finally,bioactive peptides can be isolated which alter the metabolism of theseintracellular organisms, in such a way as to terminate theirintracellular life cycle by inhibiting a key organismal event.

Antibiotic drugs that are widely used have certain dose dependent,tissue specific toxicities. For example renal toxicity is seen with theuse of gentamicin, tobramycin, and amphotericin; hepatotoxicity is seenwith the use of INH and rifampin; bone marrow toxicity is seen withchloramphenicol; and platelet toxicity is seen with ticarcillin, etc.These toxicities limit their use. Candidate libraries can be introducedinto the specific cell types where specific changes leading to cellulardamage or apoptosis by the antibiotics are produced, and bioactivepeptides can be isolated that confer protection, when these cells aretreated with these specific antibiotics.

Furthermore, the present invention finds use in screening for bioactivepeptides that block antibiotic transport mechanisms. The rapid secretionfrom the blood stream of certain antibiotics limits their usefulness.For example, penicillins are rapidly secreted by certain transportmechanisms in the kidney and choroid plexus in the brain. Probenecid isknown to block this transport and increase serum and tissue levels.Candidate agents can be inserted into specific cells derived from kidneycells and cells of the choroid plexus known to have active transportmechanisms for antibiotics. Bioactive peptides can then be isolatedwhich block the active transport of specific antibiotics and thus extendthe serum half-life of these drugs.

In a preferred embodiment, the present methods are useful in drugtoxicities and drug resistance applications. Drug toxicity is asignificant clinical problem. This may manifest itself as specifictissue or cell damage with the result that the drug's effectiveness islimited. Examples include myeloablation in high dose cancerchemotherapy, damage to epithelial cells lining the airway and gut, andhair loss. Specific examples include adriamycin induced cardiomyocytedeath, cisplatinin-induced kidney toxicity, vincristine-induced gutmotility disorders, and cyclosporin-induced kidney damage. Candidatelibraries can be introduced into specific cell types with characteristicdrug-induced phenotypic or functional responses, in the presence of thedrugs, and agents isolated which reverse or protect the specific celltype against the toxic changes when exposed to the drug. These effectsmay manifest as blocking the drug induced apoptosis of the cell ofinterest, thus initial screens will be for survival of the cells in thepresence of high levels of drugs or combinations of drugs used incombination chemotherapy.

Drug toxicity may be due to a specific metabolite produced in the liveror kidney which is highly toxic to specific cells, or as a result ofdrug interactions in the liver, block or enhance the metabolism of anadministered drug. Candidate libraries can be introduced into liver orkidney cells following the exposure of these cells to the drug known toproduce the toxic metabolite. Bioactive peptides can be isolated whichalter how the liver or kidney cells metabolize the drug, and specificagents identified which prevent the generation of a specific toxicmetabolite. The generation of the metabolite can be followed by massspectrometry, and phenotypic changes can be assessed by microscopy. Sucha screen can also be done in cultured hepatocytes, cocultured withreadout cells which are specifically sensitive to the toxic metabolite.Applications include reversible (to limit toxicity) inhibitors ofenzymes involved in drug metabolism.

Multiple drug resistance, and hence tumor cell selection, outgrowth, andrelapse, leads to morbidity and mortality in cancer patients. Candidatelibraries can be introduced into tumor cell lines (primary and cultured)that have demonstrated specific or multiple drug resistance. Bioactivepeptides can then be identified which confer drug sensitivity when thecells are exposed to the drug of interest, or to drugs used incombination chemotherapy. The readout can be the onset of apoptosis inthese cells, membrane permeability changes, the release of intracellularions and fluorescent markers. The cells in which multidrug resistanceinvolves membrane transporters can be preloaded with fluorescenttransporter substrates, and selection carried out for peptides whichblock the normal efflux of fluorescent drug from these cells. Candidatelibraries are particularly suited to screening for peptides whichreverse poorly characterized or recently discovered intracellularmechanisms of resistance or mechanisms for which few or nochemosensitizers currently exist, such as mechanisms involving LRP (lungresistance protein). This protein has been implicated in multidrugresistance in ovarian carcinoma, metastatic malignant melanoma, andacute myeloid leukemia. Particularly interesting examples includescreening for agents which reverse more than one important resistancemechanism in a single cell, which occurs in a subset of the most drugresistant cells, which are also important targets. Applications wouldinclude screening for peptide inhibitors of both MRP (multidrugresistance related protein) and LRP for treatment of resistant cells inmetastatic melanoma, for inhibitors of both p-glycoprotein and LRP inacute myeloid leukemia, and for inhibition (by any mechanism) of allthree proteins for treating pan-resistant cells.

In a preferred embodiment, the present methods are useful in improvingthe performance of existing or developmental drugs. First passmetabolism of orally administered drugs limits their oralbioavailability, and can result in diminished efficacy as well as theneed to administer more drug for a desired effect. Reversible inhibitorsof enzymes involved in first pass metabolism may thus be a usefuladjunct enhancing the efficacy of these drugs. First pass metabolismoccurs in the liver, thus inhibitors of the corresponding catabolicenzymes may enhance the effect of the cognate drugs. Reversibleinhibitors would be delivered at the same time as, or slightly before,the drug of interest. Screening of candidate libraries in hepatocytesfor inhibitors (by any mechanism, such as protein downregulation as wellas a direct inhibition of activity) of particularly problematicalisozymes would be of interest. These include the CYP3A4 isozymes ofcytochrome P450, which are involved in the first pass metabolism of theanti-HIV drugs saquinavir and indinavir. Other applications couldinclude reversible inhibitors of UDP-glucuronyltransferases,sulfotransferases, N-acetyltransferases, epoxide hydrolases, andglutathione S-transferases, depending on the drug. Screens would be donein cultured hepatocytes or liver microsomes, and could involveantibodies recognizing the specific modification performed in the liver,or co-cultured readout cells, if the metabolite had a differentbioactivity than the untransformed drug. The enzymes modifying the drugwould not necessarily have to be known, if screening was for lack ofalteration of the drug.

In a preferred embodiment, the present methods are useful inimmunobiology, inflammation, and allergic response applications.Selective regulation of T lymphocyte responses is a desired goal inorder to modulate immune-mediated diseases in a specific manner.Candidate libraries can be introduced into specific T cell subsets (TH1,TH2, CD4+, CD8+, and others) and the responses which characterize thosesubsets (cytokine generation, cytotoxicity, proliferation in response toantigen being presented by a mononuclear leukocyte, and others) modifiedby members of the library. Agents can be selected which increase ordiminish the known T cell subset physiologic response. This approachwill be useful in any number of conditions, including: 1) autoimmunediseases where one wants to induce a tolerant state (select a peptidethat inhibits T cell subset from recognizing a self-antigen bearingcell); 2) allergic diseases where one wants to decrease the stimulationof IgE producing cells (select peptide which blocks release from T cellsubsets of specific B-cell stimulating cytokines which induce switch toIgE production); 3) in transplant patients where one wants to induceselective immunosuppression (select peptide that diminishesproliferative responses of host T cells to foreign antigens); 4) inlymphoproliferative states where one wants to inhibit the growth orsensitize a specific T cell tumor to chemotherapy and/or radiation; 5)in tumor surveillance where one wants to inhibit the killing ofcytotoxic T-cells by Fas ligand bearing tumor cells; and 5) in T cellmediated inflammatory diseases such as Rheumatoid arthritis, Connectivetissue diseases (SLE), Multiple sclerosis, and inflammatory boweldisease, where one wants to inhibit the proliferation of disease-causingT cells (promote their selective apoptosis) and the resulting selectivedestruction of target tissues (cartilage, connective tissue,oligodendrocytes, gut endothelial cells, respectively).

Regulation of B cell responses will permit a more selective modulationof the type and amount of immunoglobulin made and secreted by specific Bcell subsets. Candidate libraries can be inserted into B cells andbioactive peptides selected which inhibit the release and synthesis of aspecific immunoglobulin. This may be useful in autoimmune diseasescharacterized by the overproduction of auto-antibodies and theproduction of allergy causing antibodies, such as IgE. Agents can alsobe identified which inhibit or enhance the binding of a specificimmunoglobulin subclass to a specific antigen either foreign of self.Finally, agents can be selected which inhibit the binding of a specificimmunoglobulin subclass to its receptor on specific cell types.

Similarly, agents that affect cytokine production may be selected,generally using two cell systems. For example, cytokine production frommacrophages, monocytes, etc. may be evaluated. Similarly, agents whichmimic cytokines, for example erythropoetin and IL1-17, may be selected,or agents that bind cytokines such as TNF-α, before they bind theirreceptor.

Antigen processing by mononuclear leukocytes (ML) is an important earlystep in the immune system's ability to recognize and eliminate foreignproteins. Candidate agents can be inserted into ML cell lines and agentsselected which alter the intracellular processing of foreign peptidesand sequence of the foreign peptide that is presented to T cells by MLson their cell surface in the context of Class II MHC. One can look formembers of the library that enhance immune responses of a particular Tcell subset (for example, the peptide would in fact work as a vaccine),or look for a library member that binds more tightly to MHC, thusdisplacing naturally occurring peptides, but nonetheless the agent wouldbe less immunogenic (less stimulatory to a specific T cell clone). Thisagent would in fact induce immune tolerance and/or diminish immuneresponses to foreign proteins. This approach could be used intransplantation, autoimmune diseases, and allergic diseases.

The release of inflammatory mediators (cytokines, leukotrienes,prostaglandins, platelet activating factor, histamine, neuropeptides,and other peptide and lipid mediators) is a key element in maintainingand amplifying aberrant immune responses. Candidate libraries can beinserted into MLs, mast cells, eosinophils, and other cellsparticipating in a specific inflammatory response, and bioactivepeptides selected which inhibit the synthesis, release and binding tothe cognate receptor of each of these types of mediators.

In a preferred embodiment, the present methods are useful inbiotechnology applications. Candidate library expression in mammaliancells can also be considered for other pharmaceutical-relatedapplications, such as modification of protein expression, proteinfolding, or protein secretion. One such example would be in commercialproduction of protein pharmaceuticals in CHO or other cells. Candidatelibraries resulting in bioactive peptides which select for an increasedcell growth rate (perhaps peptides mimicking growth factors or acting asagonists of growth factor signal transduction pathways), for pathogenresistance (see previous section), for lack of sialylation orglycosylation (by blocking glycotransferases or rerouting trafficking ofthe protein in the cell), for allowing growth on autoclaved media, orfor growth in serum free media, would all increase productivity anddecrease costs in the production of protein pharmaceuticals.

Random peptides displayed on the surface of circulating cells can beused as tools to identify organ, tissue, and cell specific peptidetargeting sequences. Any cell introduced into the bloodstream of ananimal expressing a library targeted to the cell surface can be selectedfor specific organ and tissue targeting. The bioactive peptide sequenceidentified can then be coupled to an antibody, enzyme, drug, imagingagent or substance for which organ targeting is desired.

Other agents which may be selected using the present inventioninclude: 1) agents which block the activity of transcription factors,using cell lines with reporter genes; 2) agents which block theinteraction of two known proteins in cells, using the absence of normalcellular functions, the mammalian two hybrid system or fluorescenceresonance energy transfer mechanisms for detection; and 3) agents may beidentified by tethering a random peptide to a protein binding region toallow interactions with molecules sterically close, i.e., within asignalling pathway, to localize the effects to a functional area ofinterest.

As previously described, and in a preferred embodiment, the presentinvention is used to identify peptides (cyclic peptides) that inhibitprotein-protein interaction. Preferred protein-protein interactions areknown protein-protein interactions. Proteins that interact in vivogenerally have physiological relevance. That is, the interaction is aresult of or functions in a regulatory pathway. Thus, by regulating theprotein-protein interaction, or inhibiting the protein-proteininteraction, the agents identified by the methods disclosed herein, mayregulate a variety of physiological pathways.

Preferred interacting proteins, including those that are involved insignal transduction pathways. By “signal transduction” is meant amechanism by which a signal is propagated between cells or within acell. Proteins involved in signal transduction are signal transductionproteins. These include, but are not limited to, proteins in the mapkinase pathway, the JAK-STAT pathway, caspace pathways involved inapoptosis, tyrosine kinase receptor signal transduction pathways,G-Protein coupled receptor signal transduction pathways, cytokinereceptor signal transaction pathways, transcriptional regulatorypathways, translational regulatory pathways, and the like.

In addition, adapter molecules involved in protein interactions can betargeted by the invention. In addition, preferred embodiments targetenzymes and substrates. That is the interaction between an enzyme and asubstrate may be prevented by the cyclic peptides disclosed herein. Thecyclic peptide may bind to the active site of an enzyme, or may bind toan allosteric regulatory site.

In a particularly preferred embodiment, the interacting proteins areinvolved in ubiquitination. Generally these molecules are referredherein as ubiquitin agents. By “ubiquitin agents” is meant a moleculeinvolved in ubiquitination. Ubiquitin agents can include ubiquitinactivating agents, ubiquitin ligating agents, and ubiquitin conjugatingagents. In addition, ubiquitin agents can include ubiquitin moieties asdescribed below. In addition, deubiquitination agents find use in theinvention. Accordingly, the methods of the invention provide methods ofincreasing ubiquitination of a target or methods for decreasingubiquitination of a target, and methods for identifying molecules thatincrease or decrease ubiquitination of a target.

Examples of ubiquitin agents are ubiquitin activating agents, ubiquitinconjugating agents, and ubiquitin ligating agents. In preferredembodiments, the ubiquitin activating agent is preferably an E1 or avariant thereof; the ubiquitin conjugating agent is preferably an E2 ora variant thereof; and the ubiquitin ligating agent is preferably an E3or variant thereof. Thus, the present invention provides methods forinhibiting the interaction of ubiquitin activating agents, ubiquitinconjugating agents, ubiquitin ligating agents, and ubiquitin moieties.In addition, the present invention provides methods of assaying foragents that modulate the attachment of an ubiquitin moiety to aubiquitin agent, target protein, or mono- or poly-ubiquitin moietypreferably attached to a ubiquitin agent or target protein.

As used herein, “ubiquitin moiety” refers to a polypeptide which istransferred or attached to another polypeptide by a ubiquitin agent.Ubiquitin moiety includes both ubiquitin and ubiquitin-like molecules.The ubiquitin moiety can comprise an ubiquitin from any species oforganism, preferably a eukaryotic species. In preferred embodiments theubiquitin moiety comprises a mammalian ubiquitin, more preferably ahuman ubiquitin. In a preferred embodiment, the ubiquitin moietycomprises a 76 amino acid human ubiquitin. In a preferred embodiment,the ubiquitin moiety comprises the amino acid sequence depicted in FIG.18. Other embodiments utilize variants of ubiquitin, as furtherdescribed below.

As used herein, “poly-ubiquitin moiety” refers to a chain of ubiquitinmoieties comprising more than one ubiquitin moiety. As used herein,“mono-ubiquitin moiety” refers to a single ubiquitin moiety. In themethods of the present invention, a mono- or poly-ubiquitin moiety canserve as a substrate molecule for the transfer or attachment ofubiquitin moiety (which can itself be a mono- or poly-ubiquitin moiety).

In a preferred embodiment, when ubiquitin moiety is attached to a targetprotein, that protein is targeted for degradation by the 26S proteasome.

As used herein, “ubiquitin moiety” encompasses naturally occurringalleles and man-made variants of ubiquitin or ubiquitin-like molecules.In a preferred embodiment the ubiquitin moiety includes a 76 amino acidpolypeptide as described in FIG. 18 or variants thereof. In a preferredembodiment, the ubiquitin moiety comprises an amino acid sequence ornucleic acid sequence corresponding to a sequence of GENBANK accessionnumber P02248, incorporated herein by reference. In other preferredembodiments, the ubiquitin moiety comprises ubiquitin-like moleculeshaving an amino acid sequence or nucleic acid sequence of a sequencecorresponding to one of the GENBANK accession numbers disclosed in TABLE4.

GENBANK accession numbers and their corresponding amino acid sequencesor nucleic acid sequences are found in the Genbank data base. Sequencescorresponding to GenBank accession numbers cited herein are incorporatedherein by reference. GenBank is known in the art, see, e.g., Benson etal., Nucleic Acids Research 26:1-7 (1998) and www.ncbi.nlm.nih.gov/.Preferably, the ubiquitin moiety has the amino acid sequence depicted inFIG. 18. In a preferred embodiment, variants of ubiquitin moiety have anoverall amino acid sequence identity of preferably greater than about75%, more preferably greater than about 80%, even more preferablygreater than about 85% and most preferably greater than 90% of the aminoacid sequence depicted in FIG. 18. In some embodiments the sequenceidentity will be as high as about 93 to 95 or 98%.

In another preferred embodiment, a ubiquitin moiety protein has anoverall sequence similarity with the amino acid sequence depicted inFIG. 18 of greater than about 80%, more preferably greater than about85%, even more preferably greater than about 90% and most preferablygreater than 93%. In some embodiments the sequence identity will be ashigh as about 95 to 98 or 99%.

As is known in the art, a number of different programs can be used toidentify whether a protein (or nucleic acid as discussed below) hassequence identity or similarity to a known sequence. Sequence identityand/or similarity is determined using standard techniques known in theart, including, but not limited to, the local sequence identityalgorithm of Smith and Waterman, Adv. Appl. Math. 2:482 (1981), by thesequence identity alignment algorithm of Needleman & Wunsch, J. Mol.Biol. 48:443 (1970), by the search for similarity method of Pearson andLipman, Proc. Natl. Acad. Sci. USA 85:2444 (1988), by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Drive, Madison, Wis.), the Best Fit sequence program describedby Devereux et al., Nucl. Acid Res. 12:387-395 (1984), preferably usingthe default settings, or by inspection. Preferably, percent identity iscalculated by FastDB based upon the following parameters: mismatchpenalty of 1; gap penalty of 1; gap size penalty of 0.33; and joiningpenalty of 30, “Current Methods in Sequence Comparison and Analysis” inMacromolecule Sequencing and Synthesis, Selected Methods andApplications, pp 127-149, Alan R. Liss, Inc. (1988).

An example of a useful algorithm is PILEUP. PILEUP creates a multiplesequence alignment from a group of related sequences using progressive,pairwise alignments. It can also plot a tree showing the clusteringrelationships used to create the alignment. PILEUP uses a simplificationof the progressive alignment method of Feng and Doolittle, J. Mol. Evol.35:351-360 (1987); the method is similar to that described by Higginsand Sharp CABIOS 5:151-153 (1989). Useful PILEUP parameters including adefault gap weight of 3.00, a default gap length weight of 0.10, andweighted end gaps.

Another example of a useful algorithm is the BLAST algorithm, describedin Altschul et al., J. Mol. Biol. 215, 403-410, (1990) and Karlin etal., Proc. Natl. Acad. Sci. USA 90:5873-5787 (1993). A particularlyuseful BLAST program is the WU-BLAST-2 program which was obtained fromAltschul et al., Methods in Enzymology, 266: 460-480 (1996);http://blast.wustl/edu/blast/README.html). WU-BLAST-2 uses severalsearch parameters, most of which are set to the default values. Theadjustable parameters are set with the following values: overlap span=1,overlap fraction=0.125, word threshold (T)=11. The HSP S and HSP S2parameters are dynamic values and are established by the program itselfdepending upon the composition of the particular sequence andcomposition of the particular database against which the sequence ofinterest is being searched; however, the values may be adjusted toincrease sensitivity.

An additional useful algorithm is gapped BLAST as reported by Altschulet al. Nucleic Acids Res. 25:3389-3402. Gapped BLAST uses BLOSUM-62substitution scores; threshold T parameter set to 9; the two-hit methodto trigger ungapped extensions; charges gap lengths of k a cost of 10+k;X_(u) set to 16, and X_(g) set to 40 for database search stage and to 67for the output stage of the algorithms. Gapped alignments are triggeredby a score corresponding to −22 bits.

A percent amino acid sequence identity value is determined by the numberof matching identical residues divided by the total number of residuesof the “longer” sequence in the aligned region. The “longer” sequence isthe one having the most actual residues in the aligned region (gapsintroduced by WU-Blast-2 to maximize the alignment score are ignored).

The alignment may include the introduction of gaps in the sequences tobe aligned. In addition, for sequences which contain either more orfewer amino acids than the amino acid sequence depicted in FIG. 18, itis understood that in one embodiment, the percentage of sequenceidentity will be determined based on the number of identical amino acidsin relation to the total number of amino acids. Thus, for example,sequence identity of sequences shorter than that of the sequencedepicted in FIG. 18, as discussed below, will be determined using thenumber of amino acids in the shorter sequence. In percent identitycalculations relative weight is not assigned to various manifestationsof sequence variation, such as, insertions, deletions, substitutions,etc.

In one embodiment, only identities are scored positively (+1) and allforms of sequence variation including gaps are assigned a value of “0”,which obviates the need for a weighted scale or parameters as describedbelow for sequence similarity calculations. Percent sequence identitycan be calculated, for example, by dividing the number of matchingidentical residues by the total number of residues of the “shorter”sequence in the aligned region and multiplying by 100. The “longer”sequence is the one having the most actual residues in the alignedregion.

Ubiquitin moieties of the present invention are polypeptides that may beshorter or longer than the amino acid sequence depicted in FIG. 18.Thus, in a preferred embodiment, included within the definition ofubiquitin moiety are portions or fragments of the amino acid sequencedepicted in FIG. 18. In one embodiment herein, fragments of ubiquitinmoiety are considered ubiquitin moieties if they are attached to anotherpolypeptide by a ubiquitin agent.

In addition, as is more fully outlined below, ubiquitin moieties of thepresent invention are polypeptides that can be made longer than theamino acid sequence depicted in FIG. 18; for example, by the addition oftags, the addition of other fusion sequences, or the elucidation ofadditional coding and non-coding sequences. As described below, thefusion of a ubiquitin moiety to a fluorescent peptide, such as GreenFluorescent Peptide (GFP), is particularly preferred.

In one embodiment, the ubiquitin moiety is an endogenous molecule. Thatis the ubiquitin moiety is naturally expressed in the cell to beassayed. However, in an alternative embodiment, the ubiquitin moiety, aswell as other proteins of the present invention, are exogenous. That is,they are recombinant proteins. A “recombinant protein” is a protein madeusing recombinant techniques, ie., through the expression of arecombinant nucleic acid as described below. In a preferred embodiment,the ubiquitin moiety of the invention is made through the expression ofa nucleic acid sequence corresponding to GENBANK accession number M26880or AB003730, or a fragment thereof. In a most preferred embodiment, thenucleic acid encodes the amino acid sequence depicted in FIG. 18.

A recombinant protein is distinguished from naturally occurring proteinby at least one or more characteristics. For example, the protein may beisolated or purified away from some or all of the proteins and compoundswith which it is normally associated in its wild type host, and thus maybe substantially pure. For example, an isolated protein is unaccompaniedby at least some of the material with which it is normally associated inits natural state, preferably constituting at least about 0.5%, morepreferably at least about 5% by weight of the total protein in a givensample. A substantially pure protein comprises at least about 75% byweight of the total protein, with at least about 80% being preferred,and at least about 90% being particularly preferred. The definitionincludes, but is not limited to, the production of a protein from oneorganism in a different organism or host cell. Alternatively, theprotein may be made at a significantly higher concentration than isnormally seen, through the use of an inducible promoter or highexpression promoter, such that the protein is made at increasedconcentration levels. Alternatively, the protein may be in a form notnormally found in nature, as in the addition of an epitope tag or aminoacid substitutions, insertions and deletions, as discussed below. In apreferred embodiment, the protein is a dominant negative as describedherein.

As used herein and further defined below, “nucleic acid” may refer toeither DNA or RNA, or molecules which contain both deoxy- andribonucleotides. The nucleic acids include genomic DNA, cDNA andoligonucleotides including sense and anti-sense nucleic acids. AlsosiRNA are included. Such nucleic acids may also contain modifications inthe ribose-phosphate backbone to increase stability and half life ofsuch molecules in physiological environments.

The nucleic acid may be double stranded, single stranded, or containportions of both double stranded or single stranded sequence. As will beappreciated by those in the art, the depiction of a single strand(“Watson”) also defines the sequence of the other strand (“Crick). Bythe term “recombinant nucleic acid” herein is meant nucleic acid,originally formed in vitro, in general, by the manipulation of nucleicacid by endonucleases, in a form not normally found in nature. Thus anisolated nucleic acid, in a linear form, or an expression vector formedin vitro by ligating DNA molecules that are not normally joined, areboth considered recombinant for the purposes of this invention. It isunderstood that once a recombinant nucleic acid is made and reintroducedinto a host cell or organism, it will replicate non-recombinantly, i.e.,using the in vivo cellular machinery of the host cell rather than invitro manipulations; however, such nucleic acids, once producedrecombinantly, although subsequently replicated non-recombinantly, arestill considered recombinant for the purposes of the invention.

The terms “polypeptide” and “protein” may be used interchangeablythroughout this application and mean at least two covalently attachedamino acids, which includes proteins, polypeptides, oligopeptides andpeptides. The protein may be made up of naturally occurring amino acidsand peptide bonds, or synthetic peptidomimetic structures. Thus “aminoacid”, or “peptide residue”, as used herein means both naturallyoccurring and synthetic amino acids. For example, homo-phenylalanine,citrulline and noreleucine are considered amino acids for the purposesof the invention. “Amino acid” also includes imino acid residues such asproline and hydroxyproline. The side chains may be in either the (R) orthe (S) configuration. In the preferred embodiment, the amino acids arein the (S) or L-configuration. If non-naturally occurring side chainsare used, non-amino acid substituents may be used, for example toprevent or retard in vivo degradation.

In one embodiment, the present invention provides compositionscontaining protein variants, for example ubiquitin moiety, E1, E2 and/orE3 variants. These variants are described in more detail in U.S. Ser.No. 10/108,767, filed Mar. 26, 2002, which is expressly incorporatedherein by reference.

In some embodiments, the interacting proteins include at least oneubiquitin activating agent. As used herein “ubiquitin activating agent”refers to a ubiquitin agent, preferably a protein, capable oftransferring or attaching a ubiquitin moiety to a ubiquitin conjugatingagent. In a preferred embodiment, the ubiquitin activating agent forms ahigh energy thiolester bond with ubiquitin moiety, thereby “activating”the ubiquitin moiety. In another preferred embodiment, the ubiquitinactivating agent binds or attaches ubiquitin moiety. In anotherpreferred embodiment, the ubiquitin activating agent is capable oftransferring or attaching ubiquitin moiety to a substrate molecule thatis a mono- or poly-ubiquitin moiety. In a preferred embodiment, theubiquitin activating agent is capable of transferring or attachingubiquitin moiety to a mono- or poly-ubiquitinated ubiquitin conjugatingagent.

In a preferred embodiment the ubiquitin activating agent is an E1. In apreferred embodiment, the E1 is capable of transferring or attachingubiquitin moiety to an E2, defined below.

In the methods and compositions of the present invention, the ubiquitinactivating agent comprises an amino acid sequence or a nucleic acidcorresponding to a sequence of an Genbank data base accession numberlisted in Table 1 below and incorporated herein by reference. TABLE 1ACCESSION ORG SYMBOL DESCRIPTION NO. Hs APPBPI amyloid beta precursorprotein NM_003905 binding protein 1, 59 kD Hs FLJ23251 hypotheticalprotein FLJ23251 NM_024818 Hs GSA7 ubiquitin activating enzyme NM_006395E1-like protein Hs similar to ubiquitin-activating XM_088743 enzyme E1(A1S9T and BN75 temperature sensitivity complementing) (H. sapiens) Hssimilar to SUMO-1 activating XM_090110 enzyme subunit 1; SUMO-1activating enzyme E1 N subunit; sentrin/SUMO- activating protein AOS1;ubiquitin-like protein SUMO-1 activating enzyme Hs SAE1 SUMO-1activating enzyme NM_005500 subunit 1 and XM_009036 Dm Uba1 Ubiquitinactivating NG_000652 enzyme 1 and NM_057962 Dm Uba2 Smt3 activatingenzyme 2 NM_080017 Hs UBA2 SUMO-1 activating enzyme NM_005499 subunit 2Hs UBE1 ubiquitin-activating enzyme NM_003334 E1 (A1S9T and BN75temperature and sensitivity complementing) XM_033895 Hs UBE1Cubiquitin-activating enzyme NM_003968 E1C (UBA3 homolog, yeast) Rn Ube1cUbiquitin-activating enzyme E1C NM_057205 Mm Ube1l Ubiquitin-activatingenzyme E1-like Hs UBE1L Ubiquitin-activating enzyme NM_003335 E1-like MmUbe1x ubiquitin-activating enzyme NM_009457 E1, Chr X Mm Ube1y1ubiquitin-activating enzyme NM_011667 E1, Chr Y 1 Mm Ube1y1-ubiquitin-activating enzyme M88481 and ps1 E1, Chr Y, pseudogene 1U09053 Mm Ube1y1- ubiquitin-activating enzyme U09054 ps2 E1, Chr Y-1,pseudogene 2

Sequences encoding a ubiquitin activating agent may also be used to makevariants thereof that are suitable for use in the methods andcompositions of the present invention. The ubiquitin activating agentsand variants suitable for use in the methods and compositions of thepresent invention may be made as described herein.

In a preferred embodiment, E1 proteins useful in the invention includethe polypeptides comprising sequence disclosed in FIGS. 19-24 orpoleptides encoded by nucleic acids having sequences disclosed in thesame figures. In other preferred embodiments, the E1 proteins areencoded by nucleic acids comprising the sequences represented by theaccession numbers provided in Table 1. In one preferred embodiment, E1is human E1. E1 is commercially available from Affiniti ResearchProducts (Exeter, U.K.). Variants of the cited E1 proteins, alsoincluded in the term “E1”, can be made as described herein.

In some embodiments, the methods of the present invention comprise theuse of a ubiquitin conjugating agent. As used herein “ubiquitinconjugating agent” refers to a ubiquitin agent, preferably a protein,capable of transferring or attaching ubiquitin moiety to a ubiquitinligating agent. In some cases, the ubiquitin conjugating agent iscapable of directly transferring or attaching ubiquitin moiety to lysineresidues in a target protein (Hershko et al., J. Biol. Chem.258:8206-8214(1983)). In a preferred embodiment, the ubiquitinconjugating agent is capable of transferring or attaching ubiquitinmoiety to a mono- or poly-ubiquitin moiety preferably attached to aubiquitin agent or target protein. In a preferred embodiment, theubiquitin conjugating agent is capable of transferring ubiquitin moietyto a mono- or poly-ubiquitinated ubiquitin ligating agent.

In a preferred embodiment the ubiquitin conjugating agent is an E2. In apreferred embodiment, ubiquitin moiety is transferred from E1 to E2. Ina preferred embodiment, the transfer results in a thiolester bond formedbetween E2 and ubiquitin moiety. In a preferred embodiment, E2 iscapable of transferring or attaching ubiquitin moiety to an E3, definedbelow.

In the methods and compositions of the present invention, the ubiquitinconjugating agent comprises an amino acid sequence or a nucleic acidsequence corresponding to a sequence of an Genbank data base accessionnumber listed in Table 2 below and incorporated herein by reference.TABLE 2 Accession No. Accession No. (nucleic acid (amino acid Name ALIASsequences) sequences) UBE2D1 Hs UBC4/5 UBE2D1, UBCH5A, UBC4/5NM_003338.1 NP_003329.1 homolog homolog UBC9 Gallus gallus UBC9,SUMO-conjugating enzyme AB069964.1 BAB68210.1 UBC9 Mus musculus mUB69U76416.1 AAB18790.1 UBC9/UBE21 Hs ?? UBE21 U45328.1 AAA86662.1 UBC9 MGC:3994, IMAGE: 2819732, BC004437.1 AAH04437.1 isoform/MGC: 3994 Hs UBC9isoform NM_003345.1 NP_003336.1 UBC9 Hs UBC9, UBE21 FTS homolog Hs+ 1aafused toes homolog, FLJ13258 NM_022476.1 NP_071921.1 FLJ13988 HsFLJ13988, clone Y79AA1002027, AK024050.1 BAB14800.1 MGC: 13396 Hs sim toE2-18 BC010900.1 AAH10900.1 UBE2V2 Hs MGC: 13396, IMAGE: 4081461NM_003350.2 NP_003341.1 MGC: 10481 Hs UBE2V2, EDAF-1, MMS2, UEV2,BC004862.1 AAH04862.1 XM_054332.1 Hs DDVIT1, ED XM_054332.1 XP_054332.1FLJ13855 Hs MGC: 10481, IMAGE: 3838157 XM_030444.3 XP_030444.1 E2-230Khomolog Hs FLJ13855 NM_022066.1 NP_071349.1 UBE2V2 Hs E2-230K ortholog,FLJ12878, NM_003339.1 NO_003330.1 UBE2D3 Hs 1 SNP KIAA1734 NM_003340.1NP_003331.1 Non-canon Ub-conj Enz UBE2D2, UBCH5B, UBC4, NM_016336.2NP_057420.2 (NCUBE1) UBC4/5 homolog NM_014176.1 NP_054895.1 HSPC150 HsUBE2D3, UBCH5C, UBC4/5 NM_016252.1 NP_057336.1 Brain 1AP repeat homologcontain 6 (BIRC6) NCUBE1, HSU93243, HSPC153, CGI-76 BIRC6, KIAA1289,apollon UBC8 Mus E2-20K, UBE2H NM_009459.1 NP_033485.1 UBC8 Hs UBE2H,UBCH, UBCH2, UBC8 NM_003344.1 NP_003335.1 UBC8 Hs 6SNP homologNM-003344.1 NP-003335.1 UBC8 Hs no 5′ UBE2H, UBCH, UBCH2, UBC8 homologRAD6 homolog Hs UBE2B, RAD6B, HHR6B, UBC2, NM_003337.1 NP_003328.1 RAD6homolog UBE2V1 var 3 Hs UBE2V1, CIR1, UEV1, UEV1A, NM_022442.2NP_071887.1 UBE2V1 var 1 Hs early CROC-1, CRO NM_021988.2 NP_068823.1stop, 56aa UBE2V1, CIR1, UEV1, UEV1A, NM_003349.3 NP_003340.1 UBE2V1 var2 Hs CROC-1, CRO UBE2V1, CIR1, UEV1, UEV1A, CROC-1, CRO UBE2L6 HsUBE2L6, UBCH8, RIG-B NM_004223.1 NP_004214.1 UBE2L3 Hs 2 SNP UBE2L3,UBCH7 NM_003347.1 NP_003338.1 UBE2E1 Hs UBE2E1, UBCH6, UBC4/5NM_003341.1 NP_003332.1 RAD6/UBE2A Hs homolog NM_003336.1 NP_003327.1UBE2E3 Hs UBE2A, RAD6A, HHR6A, UBC2, NM_006357.1 NP_006348.1 UBC12/UBE2MHs RAD6 homolog NM_003969.1 NP_003960.1 UBC7/UBE2G1 Hs UBE2E3, UBCH9,UBC4/5 NM_003342.1 NP_003333.1 homolog UBE2M, HUBC12, UBC12 homologUBE2G1, UBC7 homolog Huntingtin interact prot HIP2, LIG, E2-25KNM_005339.2 NP_005330.1 2 (HIP2) Hs LIG, HIP2 alternative splicing formABO22436.1 BAA78556.1 LIG/HIP2 variant Hs UBC6p Hs UBC6p, UBC6NM_058167.1 NP_477515.1 UBC6 Hs UBC6 AF296658.1 AAK52609.1 HBUCE1/UBE2D2var HBUCE1, LOC51619 NM_015983.1 NP_057067.1 Hs UBE2G2, UBC7 homologXM_036087.1 XP_036087.1 UBE2G2/UBC7 NCE2 NM_080678.1 NP_542409.1 homologHs CDC34, E2-CDC34, E2-32 NM_004359.1 NP_004350.1 NEDD8-conj enzyme 2complementing BC000848.1 AAH00848.1 (NCE2) Hs IMAGE: 3458173 CDC34 HsIMAGE: 3458173/NICE- 5 var UBE2C Hs UBE2C, UBCH10 NM_007019.1NP_008950.1 UBE2C possible short UBE2C, UBCH10 NM_007019.1 NP_008950.1form Hs UBC3/UBE2N Hs UBE2N, UBCH-BEN, UBC13 NM_003348.1 NP_003339.1FLJ25157 Hs hom., sim to bend AK057886.1 BAB71605.1 TSG101 Hs 1 SNPFLJ25157, highly similar to E2-23 NM_006292.1 NP_006283.1 MGC:21212/NICE-5 Tumor susceptibility gene 101 BC017708.1 AAH17708.1 var HsMCG: 21212, IMAGE: 3907760, sim to NICE-5

Sequences encoding a ubiquitin conjugating agent may also be used tomake variants thereof that are suitable for use in the methods andcompositions of the present invention. The ubiquitin conjugating agentsand variants suitable for use in the methods and compositions of thepresent invention may be made as described herein.

In a preferred embodiment, the E2 used in the methods and compositionsof the present invention comprises an amino acid sequence or nucleicacid sequence of a sequence disclosed in FIGS. 25-34 or as representedby the accession numbers in Table 2. The skilled artisan will appreciatethat many different E2 proteins and isozymes are known in the field andmay be used in the present invention, provided that the E2 has ubiquitinconjugating activity. Also specifically included within the term “E2”are variants of E2, which can be made as described herein.

In a preferred embodiment, E2 has a tag, as defined above, with thecomplex being referred to herein as “tag-E2”. Preferred E2 tags include,but are not limited to, labels, partners of binding pairs and substratebinding elements. In a most preferred embodiment, the tag is a His-tagor GST-tag.

In some embodiments, the methods of the present invention comprise theuse of a ubiquitin ligating agent. As used herein “ubiquitin ligatingagent” refers to a ubiquitin agent, preferably a protein, capable oftransferring or attaching a ubiquitin moiety to a target molecule. Insome cases, the ubiquitin agent is capable of transferring or attachingubiquitin moiety to itself or another ubiquitin ligating agent. In apreferred embodiment, the ubiquitin ligating agent is an E3.

As used herein “E3” refers to a ubiquitin ligating agent comprising oneor more subunits, preferably polypeptides, associated with the activityof E3 as a ubiquitin ligating agent (i.e., associated with the ligationor attachment of ubiquitin moiety to a target protein, and in somecases, to itself or another E3). In a preferred embodiment, E3 is amember of the HECT domain E3 ligating agents. In another preferredembodiment, E3 is a member of the RING finger domain E3 ligating agents.In a preferred embodiment, E3 comprises a ring finger subunit and aCullin subunit. Examples of RING finger polypeptides suitable for use inthe methods and compositions of the present invention include, but arenot limited to, ROC1, ROC2 and APC11. Examples of Cullin polypeptidessuitable for use in the methods and compositions of the presentinvention include, but are not limited to, CUL1, CUL2, CUL3, CUL4A,CUL4B, CUL5 and APC2. In another preferred embodiment, the E3 is mdm2,as shown in FIG. 35.

In the methods and compositions of the present invention, the ubiquitinligating agent comprises an amino acid sequence or a polypeptidesequence encoded by a nucleic acid comprising sequence corresponding toan accession number in the Genbank data base, European Molecular BiologyLaboratories (EMBL) data base, or ENSEMBL data base (a joint project ofthe European Molecular Biology Laboratories and the Sanger Institute)listed in Table 3 below and incorporated herein by reference. Theaccession numbers from the Genbank data base can be found as statedabove. The accession numbers from the EMBL data base are found atwww.embl-heidelberg.de. The accession numbers from the ENSEMBL data baseare found at www.ensembl.or. TABLE 3 Accession Accession AccessionAccession Accession Accession Accession Accession Accession No. No. No.No. No. No. No. No. No. AAD15547 AAH22038 O75485 Q96BD4 Q96K03 Q96T88Q9BYV6 Q9H073 Q9H920 AAF42995 AAH22403 O75592 Q96BD Q96K19 Q99496 Q9BZX6Q9H083 Q9H9B0 AAF91315 AAH22510 O75598 5Q96BE6 Q96K21 Q99579 Q9BZX7Q9H0A6 Q9H9B5 AAF97687 AAL30771 O75615 Q96BH1 Q96KD9 Q99675 Q9BZX8Q9H0M8 Q9H9P5 AAG50176 AAL31641 O75866 Q96BL1 Q96KL0 Q99942 Q9BZX9Q9H0V6 Q9H9T2 AAG50180 AAL36460 O76050 Q96BM5 Q96KM9 Q9BPW2 Q9BZY0Q9H0X6 Q9H9V4 AAG53500 AAL40179 O76064 Q96BQ3 Q96LD4 Q9BQ47 Q9BZY1Q9H270 Q9H9Y7 AAG53509 AAL40180 O94896 Q96BS3 Q96M70 Q9BQV0 Q9BZY2Q9H2A8 Q9HA51 AAH00832 AAL76101 O94941 Q96BX2 Q96MJ7 Q9BRZ2 Q9BZY3Q9H2S3 Q9HAC1 AAH02922 CAC81706 O94972 Q96C24 Q96MT1 Q9BS04 Q9BZY4Q9H2S4 Q9HAM2 AAH04978 CAC85986 O95159 Q96CA5 Q96MX5 Q9BSE9 Q9BZY5Q9H2S5 Q9HAP7 AAH05375 CAD19102 O95247 Q96CC2 Q96MZ7 Q9BSL8 Q9BZY6Q9H348 Q9HBD2 AAH13580 O00237 O95277 Q96D24 Q96NI4 Q9BSM1 Q9BZY8 Q9H463Q9HCL8 AAH15738 O00463 O95604 Q96D38 Q96NS4 Q9BSV9 Q9BZY9 Q9H4C2 Q9HCR0AAH16174 O00635 O95627 Q96D59 Q96NT2 KIAA066 Q9C017 Q9H4C3 Q9HCR1AAH16924 O14616 O95628 Q96DB4 Q96P09 Q9BTC5 Q9C018 Q9H4C4 Q9HCR2AAH17370 O14686 O96028 Q96DV2 Q96PF7 Q9BTD9 Q9C019 Q9H4C5 Q9HCS6AAH17585 O15057 Q14527 Q96DV3 Q96PH3 Q9BU73 Q9C021 Q9H4J2 Q9NPN4AAH17592 O15262 Q14536 Q96DX4 Q96PK3 Q9BUW4 Q9C025 Q9H5E4 Q9NPP8AAH17707 O15344 Q14848 Q96DY5 Q96PM5 Q9BUZ4 Q9C026 Q9H5F1 Q9NPQ1AAH18104 O43164 Q15156 Q96EL5 Q96PR5 Q9BV68 Q9C027 Q9H5K0 Q9NQ86AAH18107 O43255 Q15290 Q96EP1 Q96PU4 Q9BVG3 Q9C029 Q9H5L8 Q9NQP8AAH18198 O43269 Q15521 Q96EP8 Q96PX1 Q9BW41 Q9C030 Q9H5P2 Q9NR13AAH18337 O43270 Q15959 Q96EQ8 Q96QB5 Q9BW90 Q9C031 Q9H5S6 Q9NRL2AAH18647 O43567 Q16030 Q96F06 Q96QB6 Q9BWF2 Q9C032 Q9H647 Q9NRT4AAH19283 O60272 Q92550 Q96F37 Q96QY9 Q9BWL5 Q9C033 Q9H6D9 Q9NRT6AAH19355 O60291 Q92897 Q96F67 Q96RF3 Q9BWP7 Q9C034 Q9H6S6 Q9NS55AAH20556 O60372 Q969K3 Q96GF1 Q96RF8 Q9BX37 Q9C035 Q9H6W8 Q9NS56AAH20964 O60630 Q969Q1 Q96GT5 Q96RW5 Q9BXI1 Q9C036 Q9H6Y7 Q9NS56AAH20984 O75150 Q969V5 Q96H69 Q96SH4 Q9BY78 Q9C037 Q9H748 Q9NS91AAH20994 KIAA0661 Q96A37 Q96IB6 Q96SJ1 Q9BYE7 Q9C038 Q9H874 Q9NSR1AAH21258 O75162 Q96A61 Q96ID9 Q96SL3 Q9BYV2 Q9C039 Q9H890 Q9NSX7AAH21570 O75188 Q96AK4 Q96J90 Q96SR5 Q9BYV3 Q9C040 Q9H8K2 Q9NTX6AAH21571 O75341 Q96AX9 Q96JD3 Q96T06 Q9BYV4 Q9C0B0 Q9H8V9 Q9NTX7AAH21925 O75382 Q96BD3 Q96JL5 Q96T18 Q9BYV5 Q9C0G7 Q9H8W5 Q9NU68Accession Accession Accession Accession Accession Accession AccessionAccession No. No. No. No. No. No. No. No. Q9NUH2 Q9NZS9 Q9UIG0 9UQPQ7O15151 Q9BXT8 O94822 Q13263 Q9NUR4 Q9NZT8 Q9UIG1 Q9UPR2 O15541 Q9BYM8O95376 Q13489 Q9NUW5 Q9P0J9 Q9UJ97 Q9UQ11 O60858 Q9BZR9 P15918 Q13490Q9NVD5 Q9P0P0 Q9UJJ8 Q9Y225 O75678 Q9H000 P19474 Q13702 Q9NVP6 Q9P115Q9UJL3 Q9Y254 P14373 Q9NS80 P22681 Q14839 Q9NW38 Q9P1Y6 Q9UJR9 Q9Y2E6P28328 Q9NV58 P29590 Q15326 Q9NWD2 Q9P200 Q9UJV3 Q9Y2N1 P35226 Q9UDY6P35227 Q92785 Q9NWX1 Q9P2G1 Q9UKI6 Q9Y3C5 P46100 Q9UHC7 P36406 Q99728Q9NX39 Q9P2L3 Q9UKV5 Q9Y3V1 P51948 Q9ULX5 P38398 Q9HCM9 Q9NXC0 Q9P2M3Q9ULK6 Q9Y3V3 Q12899 Q9UMT8 P49754 Q9NVW2 Q9NXD0 Q9UBF6 Q9ULT6 Q9Y4I0Q12933 Q9Y4X5 P50876 Q9NYG5 Q9NXI6 Q9UDN7 Q9ULW4 Q9Y4K3 Q12986 Q9Y508P53804 Q9ULV8 Q9NZ15 Q9UEK4 Q9UMH1 Q9Y4L5 Q13049 O00623 P98170 Q9UPN9Q9NZB4 Q9UF32 Q9UMQ2 Q9Y577 Q13054 O15164 Q06587 Q9Y252 Q9NZE3 Q9UHE7Q9UNR9 Q9Y5M7 Q13114 O60683 Q12873 Q9NZE9 Q9UHW2 Q9UPQ2 Q9Y6E4 Q13434O75677 Q13191 Q9NZN6 Q9UID0 Q9UPQ4 Q9Y6U1 Q14258 O75679 Q13233Ringfinger domain Hect domain proteins proteins (Embl (GenBank data database) base) AAH19105 AAF50078 T14346 BAB23311 AAL13848 AAH19345 AAH21525NP_008944 T40821 XP_004990 AAH21144 AAH02582 S66562 NP_192994 BAB29387O00307 NP_055486 NP_008945 AAF57824 BAA92558 O00308 BAB13352 NP_032421NP_080106 AAG45422 O14996 NP_492389 AAK33088 T37964 AAF36454 O15029XP_048020 AAL39551 NP_035798 AAF36455 O15033 BAB28637 NP_175982 BAB14280AAK14420 O15036 O43165 BAA20780 AAF68076 XP_084941 BAA74919 O43584T39585 AAF68077 AAH15380 BAB24805 O94970 NP_060239 AAH11571 XP_080159BAB30794 O95071 T39007 XP_052430 AAF08298 NP_004229 O95714 BAA92539AAF68079 BAA19217 O08759 Q15386 CAC42101 AAH04712 T01491 AAH19345 Q15751XP_083009 T38951 CAB92704 NP_011374 Q96BP4 AAF79338 BAA23711 CAB09785NP_056092 Q96CZ2 NP_060382 BAB13451 NP_177189 AAH21144 Q96DE7 AAH00621AAF46512 XP_030186 NP_056986 Q96F34 AAH09271 NP_000453 AAF61856 B38919Q96F66 AAC62434 AAL29143 XP_057408 T38617 Q96GR7 AAF51314 AAL27259Q9PUN2 AAH06848 Q96J02 T21546 AAF36539 CAB99103 NP_490834 Q96PU5NP_188346 BAA84697 NP_195908 NP_010745 Q9BUI0 AAF49328 NP_499392AAH11391 CAB95249 Q9BUI6 XP_082286 AAF68080 NP_012570 Q9BVR2 NP_035020I83196 AAF52899 Q9BXZ4 NP_501120 NP_057407 AAF88143 Q9BY75 NP_055636AAF28950 AAF68614 Q9H0M0 NP_003913 XP_052223 BAA20771 Q9H2G0 BAB02722AAF68082 BAB13419 Q9H2W4 NP_497697 AAF68083 NP_011051 Q9H451 NP_490865T41750 AAH13645 Q9H783 T14761 AAH11658 Q9CUN6 Q9H9E9 AAC83345 NP_114087XP_046129 Q9HCC7 S70642 Q05086 A38920 Q9HCH9 AAG53076 T49744 AAB47756Q9NPL3 CAA03915 AAC51324 Q92462 Q9NPS9 XP_085770 BAA92571 NP_113671Q9NT88 CAC09387 BAB30733 CAA57291 Q9NWS4 NP_055421 NP_500283 XP_087357Q9NXC0 NP_523779 AAK28419 AAC41731 Q9NZS4 XP_038999 NP_446441 BAB69424Q9P0A9 AAD51453 BAA86445 T37900 Q9P2L3 AAB49301 NP_190877 T14317 Q9P2M6T49799 Q9HCE7 P51593 Q9P2P5 AAG16783 AAF50332 AAH04085 Q9UDU3 NP_195572AAH09527 BAA21482 Q9UFZ7 AAH21470 NP_490750 NP_012915 Q9UII4 NP_078878XP_003492 AAF48495 Q9ULT8 NP_073576 T37736 XP_045232 Q9Y4D8 XP_028151AAF47474 AAF50913 Q9HAU4 P46934 AAD34642 T00390 Q9HCE7 BAB28001NP_476753 P46934 NP_004658 T46412 Q05086 P46935 XP_045095 Q14669NP_524296 NP_113584 Q15034 NP_495842 AAC04845 XP_030175 1C4Z Ringfingerdomain proteins (Ensembl data base) ENSP00000259945 ENSP00000282135ENSP00000255977 ENSP00000265742 ENSP00000254436 ENSP00000280460ENSP00000283460 ENSP00000269475 ENSP00000066988 ENSP00000280461ENSP00000262370 ENSP00000265290 ENSP00000275736 ENSP00000217740ENSP00000253024 ENSP00000222597 ENSP00000275735 ENSP00000227588ENSP00000282369 ENSP00000292307 ENSP00000203439 ENSP00000259944ENSP00000253571 ENSP00000265267 ENSP00000013772 ENSP00000279757ENSP00000288913 ENSP00000263220 ENSP00000225283 ENSP00000274773ENSP00000288918 ENSP00000216225 ENSP00000246907 ENSP00000276311ENSP00000276573 ENSP00000293538 ENSP00000225285 ENSP00000166144ENSP00000237308 ENSP00000229766 ENSP00000225286 ENSP00000292363ENSP00000238203 ENSP00000242239 ENSP00000230239 ENSP00000264616ENSP00000227451 ENSP00000274616 ENSP00000286909 ENSP00000272390ENSP00000244360 ENSP00000286773 ENSP00000286910 ENSP00000272396ENSP00000244359 ENSP00000273480 ENSP00000280609 ENSP00000264767ENSP00000281105 ENSP00000217173 ENSP00000263651 ENSP00000255499ENSP00000268907 ENSP00000290337 ENSP00000261395 ENSP00000264614ENSP00000292962 ENSP00000281930 ENSP00000277584 ENSP00000262482ENSP00000280804 ENSP00000257575 ENSP00000224833 ENSP00000261481ENSP00000287546 ENSP00000287212 ENSP00000254604 ENSP00000261658ENSP00000248980 ENSP00000290788 ENSP00000240395 ENSP00000288774ENSP00000287559 ENSP00000282455 ENSP00000240318 ENSP00000261675ENSP00000264926 ENSP00000254247 ENSP00000286945 ENSP00000266880ENSP00000261737 ENSP00000290649 ENSP00000281874 ENSP00000243674ENSP00000170447 ENSP00000274542 ENSP00000240802 ENSP00000284638ENSP00000270944 ENSP00000224944 ENSP00000267825 ENSP00000247668ENSP00000289726 ENSP00000281418 ENSP00000254586 ENSP00000285317ENSP00000230099 ENSP00000289883 ENSP00000293123 ENSP00000278480ENSP00000237455 ENSP00000255325 ENSP00000285805 ENSP00000240159ENSP00000263550 ENSP00000255326 ENSP00000257633 ENSP00000294256ENSP00000264198 ENSP00000292543 ENSP00000266119 ENSP00000279766ENSP00000263464 ENSP00000277534 ENSP00000233630 ENSP00000288204ENSP00000259604 ENSP00000260947 ENSP00000264033 ENSP00000269439ENSP00000265673 ENSP00000278455 ENSP00000275619 ENSP00000268061ENSP00000248983 ENSP00000278454 ENSP00000275637 ENSP00000268058ENSP00000269391 ENSP00000274694 ENSP00000280063 ENSP00000268059ENSP00000249007 ENSP00000217740 ENSP00000276333 ENSP00000268060ENSP00000242719 ENSP00000262952 ENSP00000263651 ENSP00000261825ENSP00000217169 ENSP00000268154 ENSP00000278302 ENSP00000288587ENSP00000253642 ENSP00000265756 ENSP00000264122 ENSP00000275693ENSP00000227758 ENSP00000277490 ENSP00000284559 ENSP00000244061ENSP00000291190 ENSP00000266625 ENSP00000266252 ENSP00000272598ENSP00000261537 ENSP00000266624 ENSP00000278350 ENSP00000289818ENSP00000291733 ENSP00000258147 ENSP00000259847 ENSP00000238349ENSP00000274782 ENSP00000258148 ENSP00000274855 ENSP00000280266ENSP00000271287 ENSP00000258149 ENSP00000259930 ENSP00000242855ENSP00000261445 ENSP00000264512 ENSP00000217214 ENSP00000276688ENSP00000245836 ENSP00000261212 ENSP00000283330 ENSP00000280268ENSP00000267291 ENSP00000262642 ENSP00000263535 ENSP00000274811ENSP00000292195 ENSP00000264359 ENSP00000291416 ENSP00000268363ENSP00000216420 ENSP00000217537 ENSP00000291414 ENSP00000274828ENSP00000261464 ENSP00000264777 ENSP00000253769 ENSP00000235150ENSP00000260076 ENSP00000287880 ENSP00000274786 ENSP00000211960ENSP00000284244 ENSP00000272674 ENSP00000289896 ENSP00000262843ENSP00000292545 ENSP00000272662 ENSP00000289898 ENSP00000266952ENSP00000242669 ENSP00000293245 ENSP00000265771 ENSP00000288300ENSP00000288848 ENSP00000283875 ENSP00000229866 ENSP00000291134ENSP00000261809 ENSP00000262642 ENSP00000286475 ENSP00000261947ENSP00000262952 ENSP00000259865 ENSP00000256257 ENSP00000288715ENSP00000245937 ENSP00000217908 ENSP00000253554 ENSP00000222704ENSP00000275970 ENSP00000255004 ENSP00000259654 ENSP00000293938ENSP00000238647 ENSP00000275184 ENSP00000280266 ENSP00000266030ENSP00000268850 ENSP00000275183 ENSP00000259941 ENSP00000287335ENSP00000291963 ENSP00000200457 ENSP00000259940 ENSP00000256649ENSP00000286349 ENSP00000261537 ENSP00000270086 ENSP00000249240ENSP00000257600 ENSP00000257100 ENSP00000289140 ENSP00000253953ENSP00000281843 ENSP00000286349 ENSP00000225507 ENSP00000267073ENSP00000261245 ENSP00000252445 ENSP00000261593 ENSP00000271813ENSP00000245888 ENSP00000294213 ENSP00000257847 ENSP00000248492ENSP00000222704 ENSP00000259939 ENSP00000262881 ENSP00000265981ENSP00000245419 ENSP00000236892 ENSP00000222033 ENSP00000270280ENSP00000272023 ENSP00000238001 ENSP00000290048 ENSP00000270279ENSP00000274068 ENSP00000274657 ENSP00000274327 ENSP00000254959ENSP00000275233 ENSP00000274799

Sequences encoding a ubiquitin ligating agent may also be used to makevariants thereof that are suitable for use in the methods andcompositions of the present invention. The ubiquitin ligating agents andvariants suitable for use in the methods and compositions of the presentinvention may be made as described herein.

In a preferred embodiment, RING finger subunits include, but are notlimited to, polypeptides having an amino acid sequence corresponding toGenbank accession numbers AAD30147, AAD30146, or 6320196, incorporatedherein by reference.

In a preferred embodiment, Cullins include, but are not limited to,polypeptides having an amino acid sequence corresponding to Genbankaccession number 4503161, AAC50544, AAC36681, 4503163, AAC51190,AAD23581, 4503165, AAC36304, AAC36682, AAD45191, AAC50548, Q13620,4503167, or AAF05751, each of which is incorporated herein by reference.In addition, in the context of the invention, each of the RING fingerproteins and Cullins encompass variants of the known or listedsequences, as described herein.

These E3 ligating agents and variants may be made as described herein.In a preferred embodiment, nucleic acids used to make the RING fingerproteins include, but are not limited to, those having the nucleic acidsequences disclosed in Genbank accession numbers AF142059, AF142060 andnucleic acids 433493 to 433990 of NC 001136. In a preferred embodiment,Cullins are made from nucleic acids including, but not limited to, thosehaving nucleic acid sequences disclosed in Genbank accession numbers NM003592, U58087, AF062536, AF126404, NM 003591, U83410, NM 003590,AB014517, AF062537, AF064087, AF077188, U58091, NM 003478, X81882 andAF191337, each of which is incorporated herein by reference. Asdescribed above, variants of these sequences are also encompassed by theinvention.

In a preferred embodiment, E3 comprises the RING finger protein/Cullincombination APC11/APC2. In another preferred embodiment, E3 comprisesthe RING finger protein/Cullin combination ROC1/CUL1. In yet preferredembodiment, E3 comprises the RING finger protein/Cullin combinationROC1/CUL2. In still another preferred embodiment, E3 comprises the RINGfinger protein/Cullin combination ROC2/CUL5. However, the skilledartisan will appreciate that any combination of E3 components may beproduced and used in the invention described herein.

In an alternate embodiment, E3 comprises the ligase E3-alpha, E3A(E6-AP), HERC2, SMURF1, TRAF6, Mdm2, Cbl, Sina/Siah, Itchy, IAP orNEDD-4. In this embodiment, the ligase has the amino acid sequence ofthat disclosed in Genbank accession number AAC39845, Q05086, CAA66655,CM66654, CAA66656, AAD08657, NP_(—)002383, XP_(—)006284, AAC51970,XP_(—)013050, BAB39389, Q00987, AAF08298 or P46934, each of which isincorporated herein by reference. As above, variants are alsoencompassed by the invention. Nucleic acids for making E3 for thisembodiment include, but are not limited to, those having the sequencesdisclosed in Genbank accession numbers AF061556, XM006284, U76247,XM013050, X898032, X98031, X98033, AF071172, Z12020, AB056663, AF199364and D42055 and variants thereof.

E3 may also comprise other components, such as SKP1 and F-box proteins.The amino acid and nucleic acid sequences for SKP1 correspond to GENBANKaccession numbers AAC50241 and U33760, respectively. Many F-box proteinsare known in the art and their amino acid and nucleic acid sequences arereadily obtained by the skilled artisan from various published sources.

In a preferred embodiment, the E3 components are produced recombinantly,as described herein. In a preferred embodiment, the E3 components areco-expressed in the same host cell. Co-expression may be achieved bytransforming the cell with a vector comprising nucleic acids encodingtwo or more of the E3 components, or by transforming the host cell withseparate vectors, each comprising a single component of the desired E3protein complex. In a preferred embodiment, the RING finger protein andCullin are expressed in a single host transfected with two vectors, eachcomprising nucleic acid encoding one or the other polypeptide, asdescribed in further detail in the Examples.

In a preferred embodiment, E3 has a tag, and this complex is referred toherein as “tag-E3”. Preferably, the tag is attached to only onecomponent of the E3. Preferred E3 tags include, but are not limited to,labels, partners of binding pairs and substrate binding elements. Morepreferably, the tag is a surface substrate binding molecule. Mostpreferably, the tag is a His-tag or GST-tag. TABLE 4 Ubls ACCESSIONACCESSION NUMBERS NUMBERSS (nucleic acid (amino acid Ubl Aliassequences) sequences) Ubiquitin NM_002954.2 NP_002945 NEDD-8 NM_006156.1NP_006147 ISG-15 UCRP NM_005101.1 NP_005092.1 APG12 APG12L, NM_004707.1NP_004698.1 MAP1_LC3 APG8 MAP1_LC3, NM_022818.2 NP_073729.1 MAP1A, 1BLC3Fat10 Diubiquitin NM_006398.1 NP_006389.1 Fau, Fubi FBR-MuSV-associatedNM_001997.2 NP_001988.1 ubiquitously expressed gene, ubiquitin-likeprotein fubi, 40S ribosomal protein S30, FAU- encoded ubiquitin- likeprotein SUMO-1 Sentrin1, SMT3C, NM_003352.2 NP_003343.1 GMP1, PIC, SM,SMT3H3 SUMO-2 Sentrin3, NM_006936.1 NP_008867.1 SMT3A, SMT3H1 SUMO-3SMT3B, SMT3H2, NM_006937.2 NP_008868.2 HSMT3

Accordingly, the invention provides a method for inhibiting interactionbetween a first and a second protein. In general, any combination of aubiquitin activating, ubiquitin conjugating and ubiquitin ligating agentas well as substrates thereof can be assayed by the methods disclosedherein. That is, in one embodiment one of the proteins to be examined isa ubiquitin activating agent and the other protein is a ubiquitinconjugating agent or a ubiquitin ligating agent. Alternatively, one ofthe proteins is a ubiquitin conjugating agent and the other is aubiquitin ligating agent. Alternatively, one of the proteins is aubiquitin ligating agent and the other is a substrate of the ligase. Insome embodiments, interaction between ubiquitin conjugating agent andthe substrate also may be modulated. In a preferred embodiment, one ofthe proteins is a sub-unit of an E3 complex and the second protein alsois a sub-unit of an E3 complex.

Preferably the first protein is a ubiquitin activating agent and thesecond protein is a ubiquitin ligating agent as described above.Alternatively, the first protein is a ubiquitin ligating agent and thesecond protein is a ubiquitin conjugating agent as described above. Inaddition, the invention provides for inhibiting the interaction betweena ubiquitin conjugating agent and a substrate molecule as describedherein. In addition, the invention provides for inhibiting theinteraction between a ubiquitin ligating agent and a substrate asdescribed herein. In a preferred embodiment the ubiquitin ligating agentis mdm2 and the substrate is p53.

Alterations of protein-protein interaction may be detected by a varietyof ways as known to those of ordinary skill in the art. These will varydepending on the protein-protein interaction being examined. The methodsinclude, but are not limited to detecting a cellular readout; such as analteration in cell viability or cell cycle progression, yeast two-hybridassays, cellular FRET assays and the like. In addition, these mayinclude an alteration in enzyme activity, or production of a particularenzymatic product. In addition, co-immunoprecipitation assays may beperformed to determine an alteration in protein-protein interaction.Additional functional assays are described in U.S. application Ser. No.10/232,951, filed Aug. 30, 2002, which is expressly incorporated hereinby reference.

In a preferred embodiment inhibition of protein protein interaction isdetected by a modified yeast two-hybrid assay (see Example 3). In thisembodiment, nucleic acids encoding the interacting proteins are insertedinto the “bait” and “prey” vectors. Upon expression of the proteins inthe system, their interaction results in activation of a reporterallowing for selective cell growth. By selective cell growth is meantcell growth that is modulated by the presence of the reporter. In someembodiments the reporter allows for growth of the cells. In analternative embodiment, the reporter prevents growth of the cells. Inany event, when the cells expressing the bait and prey that interactalso express an agent that inhibits protein-protein interaction, thereporter construct is inactivated and the phenotype of the cells isreversed relative to the phenotype of the cells in the absence of theagent.

In an alternative embodiment inhibition of protein-protein interactionis detected by a FRET assay.

In a preferred embodiment, the bioactive peptide may also be used ingene therapy. In gene therapy applications, genes encoding the peptideare introduced into cells in order to achieve in vivo synthesis of atherapeutically effective genetic product. “Gene therapy” includes bothconventional gene therapy where a lasting effect is achieved by a singletreatment, and the administration of gene therapeutic agents, whichinvolves the one time or repeated administration of a therapeuticallyeffective DNA or mRNA.

There are a variety of techniques available for introducing nucleicacids into viable cells. The techniques vary depending upon whether thenucleic acid is transferred into cultured cells in vitro, or in vivo inthe cells of the intended host. Techniques suitable for the transfer ofnucleic acid into mammalian cells in vitro include the use of liposomes,electroporation, microinjection, cell fusion, DEAE-dextran, the calciumphosphate precipitation method, etc. The currently preferred in vivogene transfer techniques include transfection with viral (typicallyretroviral) vectors and viral coat protein-liposome mediatedtransfection (Dzau et al., Trends in Biotechnology 11:205-210 (1993)).In some situations it is desirable to provide the nucleic acid sourcewith an agent that targets the target cells, such as an antibodyspecific for a cell surface membrane protein or the target cell, aligand for a receptor on the target cell, etc. Where liposomes areemployed, proteins which bind to a cell surface membrane proteinassociated with endocytosis may be used for targeting and/or tofacilitate uptake, e.g., capsid proteins or fragments thereof tropic fora particular cell type, antibodies for proteins which undergointernalization in cycling, proteins that target intracellularlocalization and enhance intracellular half-life. The technique ofreceptor-mediated endocytosis is described, for example, by Wu et al.,J. Biol. Chem. 262:4429-4432 (1987); and Wagner et al., Proc. Natl.Acad. Sci. USA 87:3410-3414 (1990). For review of gene marking and genetherapy protocols see Anderson et al., Science 256:808-813 (1992).

Alternatively, an ex vivo approach can be used in which a cell excretinga therapeutically effective peptide may be transplanted into anindividual, for the constant or regulated systemic delivery of thepeptide.

The pharmaceutical compositions of the present invention comprise acompound in a form suitable for administration to a patient. In thepreferred embodiment, the pharmaceutical compositions are in a watersoluble form, such as being present as pharmaceutically acceptablesalts, which is meant to include both acid and base addition salts.“Pharmaceutically acceptable acid addition salt” refers to those saltsthat retain the biological effectiveness of the free bases and that arenot biologically or otherwise undesirable, formed with inorganic acidssuch as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid and the like, and organic acids such as acetic acid,propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid,malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid,benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid,ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid and thelike. “Pharmaceutically acceptable base addition salts” include thosederived from inorganic bases such as sodium, potassium, lithium,ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminumsalts and the like. Particularly preferred are the ammonium, potassium,sodium, calcium, and magnesium salts. Salts derived frompharmaceutically acceptable organic non-toxic bases include salts ofprimary, secondary, and tertiary amines, substituted amines includingnaturally occurring substituted amines, cyclic amines and basic ionexchange resins, such as isopropylamine, trimethylamine, diethylamine,triethylamine, tripropylamine, and ethanolamine.

The compounds can be formulated using pharmaceutically acceptablecarriers into dosages suitable for oral administration. Such carriersenable the compounds of the invention to be formulated as tablets,pills, capsules, liquids, gels, syrups, slurries, and the like for oralingestion.

The administration of the bioactive peptides of the present invention,preferably in the form of a sterile aqueous solution, can be done in avariety of ways, including, but not limited to, orally, subcutaneously,intravenously, intranasally, transdermally, intraperitoneally,intramuscularly, intrapulmonary, vaginally, rectally, or intraocularly.In some instances, for example, in the treatment of wounds,inflammation, etc., the peptide may be directly applied as a solution orspray. Depending upon the manner of introduction, the pharmaceuticalcomposition may be formulated in a variety of ways. The concentration ofthe therapeutically active peptide in the formulation may vary fromabout 0.1 to 100 weight %.

The pharmaceutical compositions may also include one or more of thefollowing: carrier proteins such as serum albumin; buffers; fillers suchas microcrystalline cellulose, lactose, corn and other starches; bindingagents; sweeteners and other flavoring agents; coloring agents; andpolyethylene glycol. Additives are well known in the art, and are usedin a variety of formulations.

The following examples serve to more fully describe the manner of usingthe above-described invention, as well as to set forth the best modescontemplated for carrying out various aspects of the invention. It isunderstood that these examples in no way serve to limit the true scopeof this invention, but rather are presented for illustrative purposes.All references cited herein are incorporated by reference.

EXAMPLES Example 1 Isolation of Inteins with Altered CyclizationActivity

A fluorescent reporter system was designed for quantifying inteincyclization. GFP was split at the loop 3 junction and the translationalorder of the N and C-terminal fragments were reversed (FIG. 12A). Thetermini were held together by a glycine-serine linker. In someconstructs, one-half of the myc epitope was fused onto either side ofthe loop 3 junction (FIG. 12A). The resulting GFP molecules werepositioned with an intein scaffold comprising either wild-type or amutant intein (FIG. 12C).

Mutant intein sequences obtained using PCR mutagenesis were screened foractivity by FACS sorting for increases in fluorescence. Western blotanalysis of several other mutants is shown in FIG. 13. In FIG. 13,several of the mutants had cyclization efficiencies greater than theparental starting intein, J3.

Example 2 Biasing a Cyclic Peptide to Reduce the Number of Conformers

To test the effects of a fixed proline in a cyclic 7 mer, theconformation space of the 7 mer cyclic peptide RGDGWS, containing twoflexible glycines was compared with that of cyclic RGPGWS using quencedmolecular dynamics calculations (O'Connor et al., J. Med. Chem.,35:2870-81 (1992); Mackay, et al., “The role of energy minimization insimulation strategies of biomolecular systems”, In Prediction of ProteinStructure and the Principles of Protein Conformation, Fasman, G., ed.,New York, Plenum Press, pp. 317-358 (1989)).

The lowest 5 kcal energy conformers were collected from a total of atleast 10,000 individual conformers obtained from multiple moleculardynamics trajectories, and compared with each other using the backboneamino acids by overlaying the structures and calculating the root meansquare deviation of these atoms in the best fit overlay using InsightII(Molecular Simulations Inc.).

An example of the cluster graph of the lowest energy conformers for eachpeptide is shown in FIGS. 16 and 17. The root mean square deviation(RMSD, Å) is coded by color, with very similar conformers (RMSD<Å) inyellow, still highly similar conformers (RMSD between 1-2 Å) in white,similar conformers (RMSD between 2-3 Å) in blue, less similar conformers(RMSD between 3-4 Å) in red, and dissimilar conformers in black (notshown).

For the cyclic peptide SRGDGWS, shown in FIG. 16 (srgdgwsLowest5A.ps),there were 62 low energy conformers. There was one family of verysimilar conformers (yellow square at bottom left) and two families ofquite similar conformers in yellow/white, one roughly in the middle ofthe graph, and one (with only moderately similar conformers) near thetop right corner. These comprised approximately 20 of the 62 conformers.The rest of the low energy conformers were not very similar to eachother, and much of the graph is red or black. Backbone overlaidconformers from most similar family, No. 1, are shown at the lower left.In the lower middle, is family No. 2. These conformers when overlaid areclearly not similar. Conformers in family No. 3 (lower right), arerather heterogeneous, although not as much as those from the red andblack regions of the graph.

For the cyclic peptide SRGPGWS, representing the substitution of pro forasp 4, the graph of the lowest energy conformers looks quite different(FIG. 17; srgpgwsLowest5B.ps). There is a much larger family of verysimilar conformers (lower left of graph, family No. 1, conformers 1-26).Family No. 2 also has very similar conformers, although they are alldifferent from family No. 1. Even family No. 3, representing over twothirds of all low energy conformers (frames 1-59) contains conformersthat are similar enough to give a blurred donut appearance. Thus,substitution of a single pro for another residue (asp in this case)clearly freezes out two additional families of conformers. As thispeptide has two glycines, the effect of proline on conformationalnarrowing of cyclic peptides with 1 or 0 glycines may be more profound.

Example 3 Two-Hybrid Assay to Detect Inhibition of Protein-ProteinInteraction

Plasmid Construction

Reporter Vector:

Full length EBNA gene coding region is amplified by polymerase chainreaction from plasmid pCEP4, purchased from Invitrogen. The EBNAfragment is then cloned into pCR2.1. Plasmid phi-EGFP is purchased fromClontech. The EBNA fragment is transferred from pCR2.1 vector into MluI-Nhe I gap of pBI-EGFP to construct pBI-R, the bi-directional doublereporter vector. A control vector pBI-EGFP-Luc is also purchased fromClontech with pBI-EGFP. This vector is used in quantitative luciferaseassay. The test and bait vectors are made in a similar manner, as willbe appreciated by those in the art. In addition, a library of inteininserts are inserted into an expression vector.

Cell Culture and Transfection

Phoenix (293 origin; see PCT/US97/01019, hereby incorporated expresslyby reference in its entirety) cells are used in all of the transientassays; however, as will be appreciated, integrated reporter constructscan be made and are generally preferable. Cells are cultured in DMEMplus 10% fetal bovine serum. Plasmids are co-transfected into cells byCa²⁺ transfection method. GFP fluorescence is visible 20 hours aftertransfection. pTBIND is a vector expression a Tet DNA binding domainonly. pVACT is a vector expressing VP16 activation domain only. pTFOS isa vector expressing Tet-FOS fusion protein. pVJUN is a vector expressionVP16-JUN fusion protein. All of the above expression vectors use CMVpromoters. 3 μg of pTBIND, or pTFOS, or pVACT or pVJUN is used intransfection of each 10 cm plate. 0.5 μg of pBI-EGFP-Luc is used intransfection of a 10 cm plate for luciferase assay. 0.5 μg of pBI-R isused in GFP color assays. Luciferase assay is done 40 hours aftertransfection using Promega luciferase assay kit and a Victor 1420multilabel counter (Wallac). Row 1 is the reporter vector pBIR only. Row2 is the reporter vector plus the bait vector pTBIND and the test vectorpVACT. Row 3 is pTFOS, pVACT and the reporter vector. Row 4 is pTFOS andpVACT and the reporter vector, co-transfected. Only Row 4 showssignificant luciferase activity above background, which indicatesFOS/JUN interaction detected by the mammalian protein interactioncloning system. Row 5 depicts luciferase activity in one of the clonesexpressing an intein construct. Luciferase activity is reduced ascompared with row 4 demonstrating that the intein prevented theinteraction of FOS/JUN. Row 6 depicts luciferase in a clone expressing adifferent intein construct and luciferase activity is not reduced,thereby demonstrating that the expression product did not interfere withFOS.JUN interaction. Luciferase Construct(s) Activity pBIR + pBIR,pTBIND, pVACT + pTFOS, pVACT, pBIR + pTFOS, pVACT, pBIR co-transfected++++ pTFOS, pVACT, pBIR, intein construct + co-transfected pTFOS, pVACT,pBIR, negative intein ++++ construct co-transfected

1-28. (canceled)
 29. A method comprising: a) contacting a culture ofcells with nucleic acid library encoding an intein-catalyzed cyclicpeptide library; b) monitoring said cells for a change in ubiquitinationof a molecule within a cell; c) isolating the nucleic acid that encodesthe cyclic peptide that when introduced into said cell caused saidchange in ubiquitination of said molecule.
 30. The method according toclaim 29, wherein said cyclic peptide inhibits ubiquitination of saidmolecule.
 31. The method according to claim 29, wherein said cyclicpeptide increases ubiquitination of said molecule.
 32. The methodaccording to claim 29, wherein said cells are mammalian cells.
 33. Themethod according to claim 29, wherein said nucleic acid librarycomprises a retroviral vector.